Association of the apolipoprotein A-IV 360 glnhis polymorphism with cerebrovascular disease obesity and depression in a Brazilian elderly population.код для вставкиСкачать
merican Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 135B:65 –68 (2005) Association of the Apolipoprotein A-IV: 360 Gln/His Polymorphism With Cerebrovascular Disease, Obesity, and Depression in a Brazilian Elderly Population T.F. Ejchel,1 L.M.Q. Araújo,1,2 L.R. Ramos,2 M.S. Cendoroglo,2 and Marı́lia de Arruda Cardoso Smith1* 1 Disciplina de Genética, Departamento de Morfologia, Universidade Federal de São Paulo, Escola Paulista de Medicina, São Paulo, SP, Brazil 2 Disciplina de Geriatria, Departamento de Clı´nica Médica, Universidade Federal de São Paulo, Escola Paulista de Medicina, São Paulo, SP, Brazil The identification of genetic polymorphisms as risk factors for complex diseases can be relevant for their prevention, diagnosis, and prognosis. The apolipoprotein A-IV: 360 Gln/His polymorphism was investigated in 383 elderly individuals, who were participants of a longitudinal study commenced in 1991. The major morbidities that affect elderly people, such as cardiovascular diseases, diabetes, low cognitive function, depression, and obesity, were extensively investigated. DNA was isolated from blood cells, amplified by PCR, and digested with Fnu4HI. In this population the frequency of the His allele was 0.056 and the genotypes were distributed according to Hardy–Weinberg equilibrium. Logistic regression analysis showed a significant association between the presence of His allele and cerebrovascular disease and/or transitory ischemic attack (odds ratio) (OR ¼ 3.070, P ¼ 0.027), obesity (OR ¼ 2.241, P ¼ 0.047), and depression (OR ¼ 2.879, P ¼ 0.005). This study indicates that the presence of the rare allele in elderly people can play a significant role in the occurrence of multifactorial diseases. This is the first study analyzing this polymorphism in elderly people in Brazil. More studies should be encouraged to elucidate the mechanisms involved in these diseases. ß 2005 Wiley-Liss, Inc. KEY WORDS: elderly population; APOA-IV: 360 polymorphism; morbidities; apolipoprotein A-IV INTRODUCTION Genetic polymorphism studies are very important to identify the difference between alleles and to search for their associa- Grant sponsor: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Grant sponsor: Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq). *Correspondence to: Marı́lia de Arruda Cardoso Smith, Ph.D., Disciplina de Genética, Departamento de Morfologia, Universidade Federal de São Paulo, Escola Paulista de Medicina, Rua Botucatu, 740, SP 04023-900, São Paulo, Brazil. E-mail: firstname.lastname@example.org Received 9 November 2004; Accepted 23 December 2004 DOI 10.1002/ajmg.b.30175 ß 2005 Wiley-Liss, Inc. tion with longevity and the most common diseases affecting elderly people, such as cardiovascular disease, diabetes, and low cognitive function. The selection of favorable genotypes and the low frequency of risk alleles can lead to a successful aging. The genetic factors that participate in lipid metabolism could be the leading key for the susceptibility or resistance to atherogenesis. It has been proposed that the isoproteins of the apolipoprotein A-IV gene (APOA-IV) may play different roles in lipids modulation [Eichner et al., 1989]. APOA-IV polymorphisms have been detected by isoeletric focusing followed by immunoblotting [Lohse et al., 1990]. DNA sequence analyses have shown that a single nucleotide substitution of a G to a T in isoform 2 converts glutamine to histidine in position 360 of the mature protein. The APOA-IV His polymorphism adds a positive charge in relation to the APOA-IV-Gln isoprotein, generating a more basic isoprotein, which is more hydrophobic and has an altered conformation. These changes may lead to more efficient protein activation and better lipoprotein binding. Despite the fact that the precise function of APOA-IV is not well known, this protein seems to participate in dietary fat absorption [Ordovas et al., 1989; Weinberg et al., 1990], in the transport of triacilglycerol [Otha et al., 1985] and in reverse transport of cholesterol [Duverger et al., 1996]. APOA-IV also appears to modulate the activity of some enzymes, such as lecithin cholesterol acyltransferase (LCAT) [Steinmetz and Utermann, 1985] and cholesteryl ester transfer protein (CETP) [Gambert et al., 1988]. APOA-IV is also found in cerebrospinal fluid (CSF) [Koch et al., 2001]. The APOA-IV genotype was associated with changes in HDL-C and LDL-C levels reflecting fat content changes in balanced diets, with individuals carrying the His allele exhibiting a favorable response [Clifton et al., 1997]. In individuals with type II diabetes, the APOA-IV Gln/His genotype was associated with a rise in risk of myocardial infarction, and obesity exacerbated this risk [Rewers et al., 1994]. A Brazilian study conducted in a general population indicated that three different variants of APOA-IV gene polymorphisms, including the 360 His variant, were associated with obesity-related traits [Fiengenbaum and Hutz, 2003]. In studies comparing centenarians and adults, and in those examining Alzheimer disease patients, elderly and general population, it has been suggested that several variants of APOA-IV polymorphisms observed in elderly people could be associated with healthier longevity [Merched et al., 1998; Pepe et al., 1998]. In our study, we derived the frequency of APOA-IV: 360 Gln/ His polymorphism in an elderly population of a community in São Paulo. We investigated the association of this polymorphism and major morbidities affecting elderly people. 66 Ejchel et al. MATERIALS AND METHODS Population Study The population that was studied consisted of participants from the Elderly Longitudinal Study [Ramos et al., 1998]. This study began in 1991 and originally involved 1,667 people over the age of 60 living in a community of São Paulo, Brazil. These subjects were clinically evaluated every 2 years and a sub sample of 383 individuals was invited to participate in our study during the 4th wave (2000–2001) of that study. We informed the participants about the study protocol and collected information about their previous medical conditions. They were evaluated by a physician and blood samples were collected for laboratory procedures. The Research Ethics Committee of UNIFESP approved this study and participants gave us informed consent according to the Helsinki Declaration. and Vernier . In brief, a 127 bp sequence containing the polymorphic site was amplified by polymerase chain reaction (PCR) using the primers HT3 (50 -CACCTGCTCCTGCTACTGCTCC-30 ) and HT5 (50 -CCTGAGGGACAAGGTCAACTC-30 ). After amplification, digestion of PCR products with Fnu4HI was performed for 3 hr at 378C. Restriction fragment length polymorphism products were resolved on a 12.5% acrilamide gel. After electrophoresis, the gels were stained with silver for fragment visualization. Gene Frequency Genotype and allele frequencies were calculated by allele counting as described by Emery . Chi-square test was applied to verify the Hardy–Weinberg equilibrium. Statistical Analyses Evaluation of Previous Morbidities * * * * * * Cardiovascular disease: individuals who reported previous myocardial infarction (MI), coronary heart disease (CHD), transitory ischemic attack (TIA), or cerebrovascular disease (CVD), or were taking specific medication prescribed by physicians. Hypertension: people using anti-hypertensive drugs or with systolic blood pressure above 140 or diastolic above 95 mmHg [adapted from Chobanian et al., 2003; WHO/ISH, 2003]. Type II diabetes: subjects taking insulin or oral medication and those that had fasting glucose equal to or above 126 mg/dl [adapted from The expert committee on the diagnosis and classification of diabetes mellitus, 1997; DECODE study group, 1999]. Obesity: characterized by a body mass index (BMI) above 27 kg/m2, as specified for persons 65 years of age and older [adapted from Rolland-Cachera et al., 1991; Lipschitz, 1994; Kyle et al., 2001]. Low cognitive function: individuals with a mini-mental state examination (MMSE) score below 24. Cognitive function was evaluated by the MMSE screening process, which was adapted to the Brazilian population taking literacy into account [Bertolucci et al., 1994a,b]. A score of less than 24 (out of 30) has 80%–90% sensitivity and 80% specificity for discriminating low cognition level from normal subjects [Tombaugh and McIntyre, 1992; Third report of NCEP, 2001]. Depression: characterized by score above 5 in a Brazilian validity version of the Older American’s Resources and Services (OARS) [adapted from Blay et al., 1988]. Although some studies showed that self-reported past history and medical records usually are concordant for selected medical conditions in the elderly [Bush et al., 1989], past history was only accepted when there was also evidence in physical exams, ECG, CT-scan, or physician’s report. DNA Extraction Total blood was collected in tubes containing 0.1% EDTA and genomic DNA was isolated using modified procedures from Lahiri and Nurnberger . Genotyping The APOA-IV: 360 Gln/His polymorphism was analyzed by procedures modified from those of Tenkanen  and Hixson Statistical analyses were performed using the SPSS 10.0 software. Allele frequencies of APOA-IV polymorphism were estimated by the gene-counting method. To test the effect of the allele in each disease we applied logistic regression using sex and age as co-variables in the models (confidence interval of 95% and significant when P < 0.05). RESULTS The frequency of His allele in this population was 0.056. Genotype distributions were according to Hardy–Weinberg equilibrium. We found 39 heterozygous and 2 homozygous individuals for this allele. The characteristics of the population are described in Table I. The genotypes were dichotomized for the presence of the His allele for statistical reasons. We found that the presence of His allele was associated with a higher frequency of CVD/TIA, depression, and obesity (P < 0.05). The other diseases listed in Table I did not show significant differences between the groups. Logistic regression analysis for each disease in relation to the presence or absence of His allele, gender, and age as covariables were performed. Diseases with significant association are described in Table II. We noticed that His allele presence increased the risk of CVD/TIA by approximately three times. His allele presence also doubled the risk of obesity. Obesity is mostly related to female sex and aging. Depression was found to be almost three times more frequent amongst His carriers. The other diseases analyzed did not show increased risk due to the presence or absence of this allele. TABLE I. Population Characteristics According to the Polymorphism of Apolipoprotein A-IV:360 Gln/His (Dichotomized for the Presence of the His Allele) N (female/male) Age in years SD CVD/TIA MI/CHD Hypertension Diabetes Low cognitive function Depression Obesity 360 Gln/Gln 360 His/þ P value 342 (234/108) 79.8 5.33 6.77% 15.81% 84.46% 64.22% 8.90% 17.98% 39.43% 41 (28/13) 79.7 5.28 17.14% 17.14% 78.05% 56.10% 0% 37.84% 58.62% 0.987 0.926 0.025* 0.781 0.293 0.308 0.052 0.005* 0.046* CVD, cerebrovascular disease; TIA, transitory ischemic attack; MI, myocardial infarction; CHD, coronary heart disease; SD, standard deviation. *Significant difference (P < 0.05). APOA-IV: 360 Polymorphism and Morbidities in an Elderly Population TABLE II. Results of the Logistic Regression Analysis for Diseases With Significant Association With the His Allele in the Population Studied (Dichotomized for the Presence of the His Allele) Disease CVD/TIA Obesity Depression Variables P OR Presence/absence of His Allele Gender (female/male) Age Presence/absence of His Allele Gender (female/male) Age Presence/absence of His Allele Gender (female/male) Age 0.027* 0.448 0.300 0.047* 0.012* 0.010* 0.005* 0.065 0.366 3.070 0.727 1.040 2.241 1.936 0.940 2.879 1.767 1.023 CVD, cerebrovascular disease; TIA, transitory ischemic attack; OR, odds ratio. *Significant difference (P < 0.05, a ¼ 0.05), confidence interval 95%. DISCUSSION We studied the APOA-IV: 360 Gln/His polymorphisms in a Brazilian elderly population ranging from 66 to 97 years old, investigating the frequency of each allele and associations with some diseases. This is the first study examining these polymorphisms and their association with the most common diseases affecting elderly people in Brazil. The majority of studies are carried out with a wider age range, but it is important to emphasize that studies conducted in an older population can demonstrate specific issues concerning the aging process. In our population the His allele frequency was 0.056 and the genotypes distribution were in accordance to Hardy–Weinberg equilibrium. We neither observed significant differences related to age and sex between the groups nor did we observe that APOA-IV Gln/His polymorphism is associated with myocardial infarction and/or coronary heart disease, hypertension, diabetes, or low cognitive function. It should be noted that there was no His carrier subject with low cognitive function in our population, which could be due to the fact that the number of His carriers is small. We found an association between His allele presence and cerebrovascular disease and/or transitory ischemic attack, obesity, and depression in our population. The mechanisms involved in these diseases could be related to differences in fatty acid metabolism in His carriers. Impaired fatty acid metabolism is involved in the occurrence of many diseases, such as depression and cardiovascular disease [Horrobin and Bennett, 1999]. The APOA-IV: 360 His isoform could have a role in brain metabolism that induces susceptibility to these diseases. Data in literature shows that APOA-IV has a role in food intake regulation in response to the presence of dietary fat as a satiety signal in the CNS [Tso et al., 2001]. APOA-IV Gln and His alleles seem to act in different manners in this response. Intervention studies have demonstrated that homozygous subjects for Gln have a better response than His carriers to weight loss treatment as a method of reducing cardiovascular disease risk, and this response is better still in diabetics [Heilbronn et al., 2000]. The lipoprotein metabolism in the brain is not well known yet, but synthesis, remodeling, and redistribution of lipids in the brain should occur in this compartment since intact particles of lipoprotein cannot cross the blood–brain barrier [Koch et al., 2001]. It is not known in which form HDL precursors are synthesized in the brain and how remodeling is done in the CSF. Some enzymes that have their activity modulated by APOA-IV, such as LCAT and CETP, mediate 67 HDL remodeling in the blood, but in the brain these pathways are still obscure. The physiological role of LCAT in vivo was determined in LCAT-deficient individuals that accumulated cholesterol in peripheral tissues and developed atherosclerosis prematurely and showed CNS alterations [Warden et al., 1989]. Some authors reported that LCAT is active in the brain and is colocalized with APOA-IV [Demeester et al., 2000]. APOA-IV and APOA-I are the most efficient activators of LCAT in plasma [Peelman et al., 1998]. The APOA-IV His isoform could activate LCAT in the brain in a way that causes a cumulative impairment of fatty acid metabolism leading to a higher prevalence of some diseases in elderly people, such as those seen in our study. There is a decrease of desaturase activity in the brain with aging [Bourre et al., 1990]. Desaturases are enzymes that participate in dietary fatty acid metabolism and regulate the formation of important mood modulating factors. Anomalies described for desaturases could have a fundamental role in the development of many multifactorial diseases, such as depression and cardiovascular disease [Horrobin and Bennett, 1999]. Aging changes combined with the presence of risk alleles in some individuals could lead to greater risk for these diseases. Our study showed that the presence of the His allele in elderly people is associated with CVD/TIA, obesity, and depression. A better understanding of this polymorphism’s role in multifactorial diseases could help in their prevention, diagnosis, and prognosis. The mechanisms involved in these associations have to be better elucidated. ACKNOWLEDGMENTS The authors are grateful to Prof. Dr. Clovis Araújo Peres and his staff for statistical analysis assistance, to the Brazilian Elderly Longitudinal Study team, to Bianca Borsatto–Galera for technical support. REFERENCES Bertolucci PF, Brucki SD, Campacci SR, Juliano Y. 1994a. The mini-mental state examination in a general population: impact of educational status. Arq Neurops 52:1–7. Bertolucci PHF, Okamoto IH, Ramos LR, Toniolo Neto J, Brucki SMD. 1994b. Consortium to establish a registry for Alzheimer’s disease. II: Bateria neuropsicológica. Arq de Neuro-psiqu 52(Suppl):101. Blay S, Ramos LR, Mari J. 1988. Validity of a Brazilian version of the older americans resources and services (OARS) mental health screening questionaire. J Am Geriat Soc 36:687–692. Bourre JM, Piciotti M, Dumont O. 1990. Delta 6 desaturase in brain and liver during development and aging. Lipids 25:354–356. Bush TL, Miller SR, Golden AL, Hale WE. 1989. Self-report and medical record report agreement of selected medical conditions in the elderly. Am J Public Health 79:1554–1556. Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo JL Jr et al. 2003. Seventh report of the Joint National Committee on prevention, detection, evaluation, and treatment of high blood pressure. Hypertension 42(6):1206–1252. Clifton P, Kind K, Jones C, Noakes M. 1997. Response to dietary fat and cholesterol and genetic polymorphisms. Clin Exp Pharmacol Physiol 24:A21–A25. DECODE study group. 1999. Glucose tolerance and mortality comparison of WHO and American diabetes association diagnostic criteria. Lancet 354:617–621. Demeester N, Castro G, Desrumaux C, De Geitere C, Fruchart JC, Santens P et al. 2000. Characterization and functional studies of lipoproteins, lipid transfer proteins, and lecithin: Cholesterol acyltransferase in CSF of normal individuals and patients with Alzheimer’s disease. J Lipid Res 41:963–974. Duverger N, Tremp G, Caillaud JM, Emmanuel F, Castro G, Fruchart JC, Steinmetz A, Denefle P. 1996. Protection against atherogenesis in mice mediated by human apolipoprotein A-IV. Science 273:966–968. 68 Ejchel et al. Eichner JE, Kuller LH, Ferrell RE, Kamboh MI. 1989. Phenotypic effects of apolipoprotein structural variation on lipid profiles. II. Apolipoprotein A-IV and quantitative lipid measures in the healthy women study. Genet Epidemiol 6:493–499. Emery AEH. 1986. Methodology in Medical Genetics-an introduction to statistical methods. Edinburgh: Longman Group Ltd. p 197. Expert panel on detection, evaluation, and treatment of high blood cholesterol in adults. 2001. Executive summary of the third report of The National Cholesterol Education Program (NCEP) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (Adult Treatment Panel III). JAMA 85(19):2486–2497. Fiengenbaum M, Hutz M. 2003. Further evidence for the association between obesity-related traits and the apolipoprotein A-IV gene. Int J Obes Relat Metab Disord 27:484–490. atherosclerosis due to deletion of a gene complex on chromosome 11. J Biol Chem 264(28):16339–16342. Otha T, Fidge NH, Nestel PJ. 1985. Studies on the in vivo and in vitro distribution of apolipoprotein A-IV in human plasma and lymph. J Clin Invest 76:1252–1260. Peelman F, Vinaimont N, Verhee A, Vanloo B, Verschelde JL, Labeur C et al. 1998. A proposed architecture for lecithin cholesterol acyl transferase (LCAT): Identification of the catalytic triad and molecular modeling. Protein Sci 7:587–599. Pepe G, Di Perna V, Resta F, Lovecchio M, Chimienti G, Colacicco AM, Capurso A. 1998. In search of a biological pattern for human longevity: Impact of apo A-IV genetic polymorphisms on lipoproteins and the hyper-Lp(a) in centenarians. Atherosclerosis 137:407–417. Gambert P, Lagrost L, Athias A, Bastiras S, Lallemant C. 1988. Role of apolipoprotein A-IV in the interconversion of HDL subclasses. Adv Exp Med Biol 243:263–269. Ramos LR, Toniolo NJ, Cendoroglo MS, Garcia JT, Najas MS, Perracini M et al. 1998. Two-year follow-up study of elderly residents in S. Paulo, Brazil: Methodology and preliminary results. Rev Saúde Pública 32: 397–407. Heilbronn LK, Noakes M, Morris AM, Kind KL, Clifton PM. 2000. 360 His polymorphism of the apolipoprotein A-IV gene and plasma lipid response to energy restricted diets in overweight subjects. Atherosclerosis 150: 187–192. Rewers M, Kamboh MI, Hoag S, Shetterly SM, Ferrell RE, Hamman RF. 1994. APOA-IV polymorphism associated with myocardial infarction in obese NIDDM patients. The San Luis valley diabetes study. Diabetes 43:1485–1489. Hixson JE, Vernier DT. 1991. Restriction isotyping of human apolipoprotein A-IV: Rapid typing of known isoforms and detection of a new isoform that deletes a conserved repeat. J Lipid Res 32:1529–1535. Rolland-Cachera MF, Cole TJ, Sempé M, Tichet JR, Rossignol C, Charraud A. 1991. Body mass index variations: Centiles from birth to 97 years. Eur J Clin Nutri 45:13–21. Horrobin DF, Bennett CN. 1999. Depression and bipolar disorder: Relationships to impaired fatty acid and phospholipid metabolism and to diabetes, cardiovascular disease, immunological abnormalities, cancer, aging, and osteoporosis. Possible candidate genes. Prostaglandins Leukot Essent Fatty Acids 60:217–234. Steinmetz A, Utermann G. 1985. Activation of lecithin: Cholesterol acyltransferase by human apolipoprotein A-IV. J Biol Chem 260:2258–2264. Koch S, Donarski N, Goetze K, Kreckel M, Stuerenburg HJ, Buhmann C, Beisiegel U. 2001. Characterization of four lipoprotein classes in human cerebrospinal fluid. J Lipid Res 42:1143–1151. Kyle UG, Genton L, Karsegard DHV, Michel J-P, Slosman DO, Pichard C. 2001. Total body mass, fat mass, fat-free mass, and skeletal differences in 60-year-old persons. J Am Geriat Soc 49:1633–1640. Lahiri DK, Nurnberger JI. 1991. A rapid non-enzymatic method for the preparation of HMW DNA from blood for RFLP studies. Nucleic Acids Res 19:5444. Lipschitz DA. 1994. Screening for nutritional status in the elderly. Prim Care 21(1):55–66. Lohse P, Kindt MR, Rader DJ, Brewer HB Jr. 1990. Genetic polymorphism of human plasma apolipoprotein A-IV is due to nucleotide substitutions in the apolipoprotein A-IV gene. J Biol Chem 265:10061–10064. Merched A, Xia Y, Papadopoulou A, Siest G, Visvikis S. 1998. Apolipoprotein AIV codon 360 mutation increases with human aging and is not associated with Alzheimer’s disease. Neurosci Lett 242:117–119. Ordovas JM, Cassidy DK, Civeira F, Bisgaier CL, Schaefer EJ. 1989. Familial apolipoprotein A-I, C-III, and A-IV deficiency and premature Tenkanen H. 1989. Genotyping of apolipoprotein A-IV by digestion of amplified DNA with restriction endonuclease Fnu4H1: Use of tailored primer to abolish additional recognition sites during the gene amplification. J Lipid Res 30:545–549. The expert committee on the diagnosis and classification of diabetes mellitus. 1997. Report of the expert committee on the diagnosis and classification of Diabetes Mellitus. Diabetes Core 20:90–96. Tombaugh TN, McIntyre NJ. 1992. The mini-mental state examination: A comprehensive review. J Am Geriat Soc 40:922–935. Tso P, Liu M, Kalogeris TJ, Thomson AB. 2001. The role of apolipoprotein A-IV in the regulation of food intake. Annu Rev Nutr 21:231–254. Warden CH, Langner CA, Gordon JI, Taylor BA, McLean JW, Lusis AJ. 1989. Tissue-specific expression, developmental regulation, and chromosomal mapping of the lecithin: Cholesterol acyltransferase gene. Evidence for expression in brain and testes as well as liver. J Biol Chem 264:21573–21581. Weinberg RB, Dantzker C, Patton CS. 1990. Sensitivity of serum apolipoprotein A-IV levels to changes in dietary fat content. Gastroenterology 98:17–24 WHO/ISH writing group. 2003. WHO/ISH, World Health Organization (WHO)/International Society of Hypertension (ISH) statement on management of hypertension. J Hypertension 21:1983–1992.