Genetic evidence that apolipoprotein E4 is not a relevant susceptibility factor for cholelithiasis in two high-risk populations
2007; Elsevier BV; Volume: 48; Issue: 6 Linguagem: Inglês
10.1194/jlr.m700059-jlr200
ISSN1539-7262
AutoresJuan G. Mella, Ramin Schirin-Sokhan, Attilio Rigotti, Fernando Pimentel, Luís Villarroel, Hermann E. Wasmuth, Tilman Sauerbruch, Flavio Nervi, Frank Lammert, Juan Francisco Miquel,
Tópico(s)Phytase and its Applications
ResumoApolipoprotein E (apoE) isoforms are genetic determinants of interindividual variations in lipid metabolism. To assess whether apoE is a genetic risk factor for cholesterol gallstone disease (GD), we analyzed apoE variants in populations from Chile and Germany, two countries with very high prevalence rates of this disease. ApoE genotypes were determined in Chilean gallstone patients (n = 117) and control subjects (n = 122) as well as in German gallstone patients (n = 184) and matched controls (n = 184). In addition, we studied apoE variants in subgroups of Chilean patients with strong differences in their susceptibility to acquire gallstones: 50 elderly subjects without gallstones in spite of well-known risk factors for this disease (gallstone-resistant) and 32 young individuals with gallstones but without risk factors (gallstone-susceptible). Furthermore, correlation analysis of apoE genotypes with cholesterol crystal formation times, biliary cholesterol saturation index (CSI), and gallstone cholesterol contents was performed in 81 cholecystectomized patients. In this study analyzing the largest sample set available, apoE4 genotype was not associated with an increased frequency of GD in either population. Moreover, in the Chilean population after adjusting for risk factors such as gender, age, body mass index, serum lipids, and glucose, the odds ratio for the association of the apoE4 allele and GD was significantly (P < 0.05) <1. Also, genotypes were not correlated with cholesterol crystal formation time, CSI, or gallstone cholesterol content. In contrast to previous smaller studies, apoE polymorphisms were not associated with susceptibility to cholesterol GD in high-risk populations. Apolipoprotein E (apoE) isoforms are genetic determinants of interindividual variations in lipid metabolism. To assess whether apoE is a genetic risk factor for cholesterol gallstone disease (GD), we analyzed apoE variants in populations from Chile and Germany, two countries with very high prevalence rates of this disease. ApoE genotypes were determined in Chilean gallstone patients (n = 117) and control subjects (n = 122) as well as in German gallstone patients (n = 184) and matched controls (n = 184). In addition, we studied apoE variants in subgroups of Chilean patients with strong differences in their susceptibility to acquire gallstones: 50 elderly subjects without gallstones in spite of well-known risk factors for this disease (gallstone-resistant) and 32 young individuals with gallstones but without risk factors (gallstone-susceptible). Furthermore, correlation analysis of apoE genotypes with cholesterol crystal formation times, biliary cholesterol saturation index (CSI), and gallstone cholesterol contents was performed in 81 cholecystectomized patients. In this study analyzing the largest sample set available, apoE4 genotype was not associated with an increased frequency of GD in either population. Moreover, in the Chilean population after adjusting for risk factors such as gender, age, body mass index, serum lipids, and glucose, the odds ratio for the association of the apoE4 allele and GD was significantly (P < 0.05) <1. Also, genotypes were not correlated with cholesterol crystal formation time, CSI, or gallstone cholesterol content. In contrast to previous smaller studies, apoE polymorphisms were not associated with susceptibility to cholesterol GD in high-risk populations. apolipoprotein E body mass index cholesterol saturation index gallstone disease Although the precise cause of cholelithiasis is unknown, several lines of evidence have indicated that it is a multifactorial disease in which genetic and environmental factors are involved (1.Portincasa P. Moschetta A. Palasciano G. 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Genetic Basis of Common Diseases. Oxford University Press, New York2002: 298-335Google Scholar, 3.Carey M.C. Pathogenesis of gallstones.Am. J. Surg. 1993; 165: 410-419Abstract Full Text PDF PubMed Scopus (140) Google Scholar, 4.Lammert F. Sauerbruch T. Mechanisms of disease: the genetic epidemiology of gallbladder stones.Nat. Clin. Pract. Gastroenterol. Hepatol. 2005; 2: 423-433Crossref PubMed Scopus (129) Google Scholar, 17.Busch N. Lammert F. Matern S. Biliary secretory immunoglobulin A is a major constituent of the new group of cholesterol crystal-binding proteins.Gastroenterology. 1998; 115: 129-138Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 18.Busch N. Lammert F. Marschall H.U. Matern S. A new subgroup of lectin-bound biliary proteins binds to cholesterol crystals, modifies crystal morphology, and inhibits cholesterol crystallization.J. Clin. Invest. 1995; 96: 3009-3015Crossref PubMed Scopus (29) Google Scholar, 19.Miquel J.F. Nunez L. Rigotti A. Amigo L. Brandan E. Nervi F. Isolation and partial characterization of cholesterol pronucleating hydrophobic glycoproteins associated to native biliary vesicles.FEBS Lett. 1993; 318: 45-49Crossref PubMed Scopus (19) Google Scholar). Together, these findings suggest that abnormalities in cholesterol and lipoprotein metabolism play an important role in the pathogenesis of cholesterol cholelithiasis. Apolipoprotein E (apoE) plays a central role in the overall regulation of cholesterol metabolism (20.Mahley R.W. Apolipoprotein E: cholesterol transport protein with expanding role in cell biology.Science. 1988; 240: 622-630Crossref PubMed Scopus (3383) Google Scholar, 21.Miettinen T.A. Impact of apo E phenotype on the regulation of cholesterol metabolism.Ann. Med. 1991; 23: 181-186Crossref PubMed Scopus (52) Google Scholar). 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Res. 1988; 20: 26-31PubMed Google Scholar). Moreover, it has been demonstrated that the apoE polymorphism is associated with an increased risk for coronary heart disease and Alzheimer disease (26.Tiret L. Knijff P.de Menzel H.J. Ehnholm C. Nicaud V. Havekes L.M. ApoE polymorphism and predisposition to coronary heart disease in youths of different European populations. The EARS Study. European Atherosclerosis Research Study.Arterioscler. Thromb. 1994; 14: 1617-1624Crossref PubMed Google Scholar, 27.Evans R.M. Hui S. Perkins A. Lahiri D.K. Poirier J. Farlow M.R. Cholesterol and APOE genotype interact to influence Alzheimer disease progression.Neurology. 2004; 62: 1869-1871Crossref PubMed Scopus (86) Google Scholar). Two previous studies suggested an association of apoE with cholesterol GD: the apoE4 isoform was associated with increased gallstone cholesterol content in cholecystectomized patients in Finland (28.Juvonen T. Kervinen K. Kairaluoma M.I. Lajunen L.H. Kesaniemi Y.A. Gallstone cholesterol content is related to apolipoprotein E polymorphism.Gastroenterology. 1993; 104: 1806-1813Abstract Full Text PDF PubMed Scopus (72) Google Scholar) and with a higher risk for gallstones in a case-control study in Spain (29.Bertomeu A. Ros E. Zambon D. Vela M. Perez-Ayuso R.M. Targarona E. Trias M. Sanllehy C. Casals E. Ribo J.M. Apolipoprotein E polymorphism and gallstones.Gastroenterology. 1996; 111: 1603-1610Abstract Full Text PDF PubMed Scopus (97) Google Scholar). The apoE2 isoform, by contrast, was found less frequently in Finnish women with gallstones than in controls (30.Niemi M. Kervinen K. Rantala A. Kauma H. Paivansalo M. Savolainen M.J. Lilja M. Kesaniemi Y.A. The role of apolipoprotein E and glucose intolerance in gallstone disease in middle aged subjects.Gut. 1999; 44: 557-562Crossref PubMed Scopus (35) Google Scholar). However, other studies have not yielded consistent association findings (31.Lin Q.Y. Du J.P. Zhang M.Y. Yao Y.G. Li L. Cheng N.S. 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Invest. 1990; 20: 143-149Crossref PubMed Scopus (14) Google Scholar). This would explain the reduction of fecal bile salt excretion observed in apoE4 carriers (35.Fischer S. Dolu M.H. Zundt B. Meyer G. Geisler S. Jungst D. Apolipoprotein E polymorphism and lithogenic factors in gallbladder bile.Eur. J. Clin. Invest. 2001; 31: 789-795Crossref PubMed Scopus (23) Google Scholar) and lead to a decreased bile salt-cholesterol ratio in bile, although no significant difference in cholesterol saturation of gallbladder bile from patients with different apoE isoforms has been reported (28.Juvonen T. Kervinen K. Kairaluoma M.I. Lajunen L.H. Kesaniemi Y.A. Gallstone cholesterol content is related to apolipoprotein E polymorphism.Gastroenterology. 1993; 104: 1806-1813Abstract Full Text PDF PubMed Scopus (72) Google Scholar, 32.Hasegawa K. Terada S. Kubota K. Itakura H. Imamura H. Ohnishi S. Aoki T. Ijichi M. Saiura A. Makuuchi M. Effect of apolipoprotein E polymorphism on bile lipid composition and the formation of cholesterol gallstone.Am. J. Gastroenterol. 2003; 98: 1605-1609Crossref PubMed Scopus (19) Google Scholar, 38.Angelin B. Holmquist L. Leijd B. Einarsson K. Bile acid metabolism in familial dysbetalipoproteinaemia: studies in subjects with the apolipoprotein E-2/2 phenotype.Eur. J. Clin. Invest. 1990; 20: 143-149Crossref PubMed Scopus (14) Google Scholar, 39.Van Erpecum K.J. Van Berge-Henegouwen G.P. Eckhardt E.R. Portincasa P. Van De Heijning B.J. Dallinga-Thie G.M. Groen A.K. Cholesterol crystallization in human gallbladder bile: relation to gallstone number, bile composition, and apolipoprotein E4 isoform.Hepatology. 1998; 27: 1508-1516Crossref PubMed Scopus (27) Google Scholar). Because holo-apoE is present in bile, it might alternatively, similar to apoA-I and apoA-II, have a role in the destabilization of bile and the modulation of cholesterol crystallization and stone growth. To clarify whether apoE variants modulate susceptibility to GD, we performed a large combined genetic association (case-control) study in Chile and Germany, two countries with high-risk populations for cholesterol GD (8.Miquel J.F. Covarrubias C. Villaroel L. Mingrone G. Greco A.V. Puglielli L. Carvallo P. Marshall G. Pino G.Del Nervi F. Genetic epidemiology of cholesterol cholelithiasis among Chilean Hispanics, Amerindians, and Maoris.Gastroenterology. 1998; 115: 937-946Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 40.Marinovic I. Guerra C. Larach G. Incidence of cholelithiasis in autopsy material and analysis of the composition of calculi. [In Spanish.].Rev. Med. Chil. 1972; 100: 1320-1327PubMed Google Scholar, 41.Volzke H. Baumeister S.E. Alte D. Hoffmann W. Schwahn C. Simon P. John U. Lerch M.M. Independent risk factors for gallstone formation in a region with high cholelithiasis prevalence.Digestion. 2005; 71: 97-105Crossref PubMed Scopus (177) Google Scholar). In addition, we determined apoE polymorphisms among two selected groups of patients with extreme differences in their susceptibility to acquire gallstones: old subjects without gallstones in spite of well-known risk factors for GD and young gallstone carriers without known risk factors (8.Miquel J.F. Covarrubias C. Villaroel L. Mingrone G. Greco A.V. Puglielli L. Carvallo P. Marshall G. Pino G.Del Nervi F. Genetic epidemiology of cholesterol cholelithiasis among Chilean Hispanics, Amerindians, and Maoris.Gastroenterology. 1998; 115: 937-946Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 42.Amigo L. Zanlungo S. Mendoza H. Miquel J.F. Nervi F. Risk factors and pathogenesis of cholesterol gallstones: state of the art.Eur. Rev. Med. Pharmacol. Sci. 1999; 3: 241-246PubMed Google Scholar, 43.Maurer K.R. Everhart J.E. Knowler W.C. Shawker T.H. Roth H.P. Risk factors for gallstone disease in the Hispanic populations of the United States.Am. J. Epidemiol. 1990; 131: 836-844Crossref PubMed Scopus (103) Google Scholar). Finally, to assess whether apoE polymorphisms influence bile lithogenicity, we correlated apoE genotypes and cholesterol crystal formation time, biliary cholesterol saturation index (CSI), and gallstone cholesterol content in Chilean patients subjected to cholecystectomy. Chilean patients included in this study represent a nested case-control study based on an ongoing population-based study of the prevalence and risk factors of cholelithiasis in Chile (8.Miquel J.F. Covarrubias C. Villaroel L. Mingrone G. Greco A.V. Puglielli L. Carvallo P. Marshall G. Pino G.Del Nervi F. Genetic epidemiology of cholesterol cholelithiasis among Chilean Hispanics, Amerindians, and Maoris.Gastroenterology. 1998; 115: 937-946Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). The survey was performed in La Florida, an urban area of Santiago, which is representative of the predominant socioeconomic strata and European-Amerindian admixture of the Chilean population. We randomly sampled 2,558 adult individuals aged 18 years and older, and 1,678 subjects (66%) answered a questionnaire and attended a general health examination including abdominal ultrasonography. For the genetic study, 250 unrelated individuals [median age of 44 years (range, 18–82 years); 66 males, 168 females] with (n = 125) and without (n = 125) GD were randomly selected from the adult population described above. Eleven subjects were excluded because DNA was not available or PCR for apoE genotyping did not generate a product. No subjects received lipid-lowering drugs, and familial hypercholesterolemia was not detected in this cohort. The German patients were recruited from 747 inpatients aged 18 years and older at the Department of Medicine III and the Department of Surgery, University Hospital Aachen. All patients admitted to the gastroenterology ward of the Department of Medicine III were included consecutively to exclude potential bias, and gallstone status and clinical chemical parameters were obtained on the day after admission to minimize the effects of the underlying diseases on liver metabolism and gallbladder stasis. No subjects received lipid-lowering drugs, and patients with known familial hypercholesterolemia were excluded. In the Department of Surgery, we included patients undergoing elective laparoscopic cholecystectomy only. Overall, 368 Caucasian patients (184 cases, 184 matched controls) with a median age of 64 years (range, 30–89 years) were enrolled (162 males, 206 females). GD cases were defined as subjects who had a previous cholecystectomy for gallstones or presented cholecystolithiasis at the time of the study, as confirmed by ultrasound. Abdominal ultrasonography was performed by three trained operators, unaware of the subject's medical history, using real-time machines with 3.5 MHz linear transducers. The presence of gallstones was assessed according to well-accepted ultrasonographic criteria (7.Barbara L. Sama C. Morselli Labate A.M. Taroni F. Rusticali A.G. Festi D. Sapio C. Roda E. Banterle C. Puci A. et al.A population study on the prevalence of gallstone disease: the Sirmione Study.Hepatology. 1987; 7: 913-917Crossref PubMed Scopus (504) Google Scholar). Blood samples were obtained from fasted individuals for chemical analysis and DNA extraction. To increase the chance of finding potential genetic factors involved in gallstone formation, we compared apoE genotype frequencies in two additional subgroups of Chilean patients with strong differences in their genetic susceptibility to this disease. For that purpose, we used the following selection criteria: i) gallstone-susceptible individuals were <30 years old, nonobese [body mass index (BMI) < 27 kg/m2], nonmultiparous female or male patients with gallstones (n = 32); ii) gallstone-resistant individuals were >50 years old multiparous female or >60 years old male patients, obese (BMI > 27 kg/m2), without gallstones (n = 50). In addition, apoE polymorphism, gallstone cholesterol content, CSI, and cholesterol crystal formation time of gallbladder bile were investigated in a group of consecutive patients with cholesterol gallstones subjected to cholecystectomy at the Pontificia Universidad Católica Clinic in Santiago (n = 81). The studies were approved by the respective institutional ethical committees. Informed consent was obtained from all subjects. ApoE genotyping was performed by PCR-restriction fragment length polymorphism analysis as described by Hixson and Vernier (44.Hixson J.E. Vernier D.T. Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with HhaI.J. Lipid Res. 1990; 31: 545-548Abstract Full Text PDF PubMed Google Scholar). We decided to use an apoE genotyping method instead of any of the apoE phenotyping methods to avoid common misclassifications, particularly of E2/E3 isoforms (45.Dallinga-Thie G.M. van Linde-Sibenius Trip M. Kock L.A. De Bruin T.W. Apolipoprotein E2/E3/E4 genotyping with agarose gels.Clin. Chem. 1995; 41: 73-75Crossref PubMed Scopus (50) Google Scholar). In brief, DNA was extracted from whole blood samples (46.Lahiri D.K. Nurnberger Jr, J.I. A rapid non-enzymatic method for the preparation of HMW DNA from blood for RFLP studies.Nucleic Acids Res. 1991; 19: 5444Crossref PubMed Scopus (1970) Google Scholar), and apoE sequences that encompass amino acid positions 112 and 158 were amplified by PCR using primers 5′-ACAGAATTCGCCCCGGCCTGGTACAC-3′ and 5′-TAAGCTTGGCACGGCTGTCCAAGGA-3′ (47.Emi M. Wu L.L. Robertson M.A. Myers R.L. Hegele R.A. Williams R.R. White R. Lalouel J.M. Genotyping and sequence analysis of apolipoprotein E isoforms.Genomics. 1988; 3: 373-379Crossref PubMed Scopus (265) Google Scholar). The 244 bp amplification product was digested with HhaI and subjected to electrophoresis on 10% polyacrylamide gels. ApoE genotypes were distinguished by the recognition of unique combinations of HhaI fragment sizes (44.Hixson J.E. Vernier D.T. Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with HhaI.J. Lipid Res. 1990; 31: 545-548Abstract Full Text PDF PubMed Google Scholar). Gallbladder bile was obtained from 81 consecutive symptomatic gallstone patients at the beginning of the cholecystectomy by needle aspiration of the gallbladder. Care was taken to avoid blood contamination of bile and to obtain a complete aspiration of the gallbladder bile to avoid the effect of stratification (48.Nunez L. Amigo L. Mingrone G. Rigotti A. Puglielli L. Raddatz A. Pimentel F. Greco A.V. Gonzalez S. Garrido J. et al.Biliary aminopeptidase-N and the cholesterol crystallisation defect in cholelithiasis.Gut. 1995; 37: 422-426Crossref PubMed Scopus (15) Google Scholar). Bile was collected in sterile tubes containing 0.05% chloramphenicol, 3 mM sodium azide, 0.2 mM thiomersal, 1 mM phenylmethylsulfonyl fluoride, and 1 mM leupeptin as preservatives. Two milliliters of bile were immediately processed for the crystal formation time assay, and the remaining bile was stored at −20°C. Gallbladder stones were washed with distilled water, dried at 37°C for 5 days, and crushed thoroughly. Their cholesterol content was subsequently determined chemically (49.Fromm H. Amin P. Klein H. Kupke I. Use of a simple enzymatic assay for cholesterol analysis in human bile.J. Lipid Res. 1980; 21: 259-261Abstract Full Text PDF PubMed Google Scholar). Only sterile bile specimens were included in the analysis of the data. Biliary cholesterol, bile salts, and phospholipids were quantified by standard techniques (50.Miquel J.F. Nunez L. Amigo L. Gonzalez S. Raddatz A. Rigotti A. Nervi F. Cholesterol saturation, not proteins or cholecystitis, is critical for crystal formation in human gallbladder bile.Gastroenterology. 1998; 114: 1016-1023Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). CSI was calculated according to Carey's critical tables (51.Carey M.C. Critical tables for calculating the cholesterol saturation of native bile.J. Lipid Res. 1978; 19: 945-955Abstract Full Text PDF PubMed Google Scholar). Serum triglyceride, total cholesterol, LDL cholesterol, and HDL cholesterol levels were determined by standard enzymatic assays (Flex® reagent cartridges; Dade Behring, Inc., Newark, DE) on the Dimension® clinical chemistry system. For the cholesterol crystal formation time measurement, aliquots (500 μl) of isotropic gallbladder bile were filtered through a sterile 0.22 μm filter (Micro Filtration Systems, Dublin, CA), flushed with N2, and incubated at 37°C in vials covered with Teflon-lined screw caps. The cholesterol crystal formation time represents the interval (in days) between time zero and the first appearance of plate-like cholesterol monohydrate crystals (50.Miquel J.F. Nunez L. Amigo L. Gonzalez S. Raddatz A. Rigotti A. Nervi F. Cholesterol saturation, not proteins or cholecystitis, is critical for crystal formation in human gallbladder bile.Gastroenterology. 1998; 114: 1016-1023Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The samples were observed for a maximum period of 21 days. When cholesterol crystal
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