Apolipoprotein E (ApoE) is a plasma protein that plays a prominent role in lipid metabolism and cholesterol transport. The gene is polymorphic: three alleles, ε2, ε3, and ε4 code for three different protein isoforms, E2, E3, and E4 respectively, each of which has different genetic implications. Carriers of ε2 and ε4 alleles have shown greater susceptibility to diseases such as lipid metabolism problems, cardiovascular disease, and Alzheimer’s disease. Although ε4 is the ancestral form of the gene, the most common allele in the human population currently is the ε3 allele. The three isoforms of ApoE generally occur in all populations at different frequencies. However, the frequencies of the various alleles have not been examined in the Midwest. The purpose of this project was to determine if a correlation existed between frequencies of ApoE mutation and heritage in Midwest individuals by using real-time PCR. It was hypothesized that this method could be an alternative and cost effective method to sequence an individual’s genome for personalized healthcare purposes. In 2000, Dr. Vann and her research assistants collected saliva samples from approximately 300 anonymous volunteers participating in the UniverCity fair hosted by Ball State University. The samples were catalogued in an Excel database with their corresponding information (gender, self- reported lineage). From this database, 50 samples were randomly selected for this study and the DNA was screened by real-time PCR for isoforms of ApoE. A handful of individuals with altered alleles were identified. The specific number of each isoform and the genotype of each individual were determined. Only one of the 50 individuals resulted in a non-wild type haplotype. A confirmatory melt curve analysis of this major heritage group A individual resulted in a homozygous E4 genotype. Unfortunately, a correlation between frequencies of ApoE mutation and heritage in Midwest individuals could not be inferred based on one differing individual. Thus, we did not have sufficient non-wild type haplotypes to permit us to amplify the variable regions of maternally inherited mitochondrial DNA for sequencing. Those sequences could have been aligned with the Cambridge Reference Sequence (Mitomap 2006) to determine maternal lineage or haplotype, which could then be correlated with self-reported lineage, and the presence of specific isoforms of ApoE. / Department of Biology

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Estudos demonstram a associação de alterações da apolipoproteína E (ApoE) e do receptor do LDL (RLDL) com a ocorrência de doenças cardiovasculares e dislipidemia. O objetivo principal deste trabalho foi investigar a associação entre genótipos diferenciais da ApoE e do RLDL com a persistência de alterações de variáveis lipídicas em indivíduos jovens acompanhados há 28 anos no Estudo do Rio de Janeiro (ERJ). Através de um estudo longitudinal, tipo coorte, investigou-se 56 indivíduos (35M) em três avaliações. Em A1 (13.301.53 anos), A2 (22.091.91 anos) e A3 (31.231.99). Nas três ocasiões foi realizada avaliação clínica. Em A2 e A3 foram dosados colesterol total, HDL, LDL e triglicerídeos. Em A3 acrescentou-se o estudo dos polimorfismos genéticos da ApoE e do RLDL. Os fragmentos de interesse neste estudo foram amplificados por PCR (polymerase chain reaction) e os genótipos foram identificados através de reações de restrição. As frequências genotípicas de ApoE foram ε3/ε3 (62,5%), ε3/ε4 (25%), ε2/ε3 (5,4%) ,ε2/ε4 (5,4%) e ε4/ε4 (1,8%) e para os genótipos de RLDL foram AA (85,7%), AT (12,5%) e TT (1,8%). O genótipo ε2/ε2 não foi observado. A análise da distribuição dos genótipos de ApoE segundo a permanência de dislipidemia mostrou que todos os indivíduos com genótipo de ApoE dos tipos ε2/ε4 e ε4/ε4 mantiveram pelo menos um lípide alterado em A2 e A3 entretanto, todos os indivíduos com genótipo de ApoE do tipo ε2/ε3 não apresentaram lípides alterados em A2 e A3. Para o genótipo do RLDL não houve diferença significativa. Quando analisadas isoladamente, não foi identificado nenhum resultado significativo em A2 e/ou A3 associado a estes genótipos. O polimorfismo do gene da ApoE esteve associado à permanência de dislipidemia em indivíduos jovens acompanhados em estudo de seguimento longitudinal / Studies have shown the association of changes in the apolipoprotein E (ApoE) and LDL receptor (LDLR) with the occurrence of cardiovascular diseases and dyslipidemia. The objective of this study was to investigate the association between different ApoE genotypes and LDLR with the persistence of changes in lipid variables in young individuals followed-up 28 years in the study of Rio de Janeiro (ERJ). Through a longitudinal study cohort, 56 subjects (35M) in A1(13.30 1.53 years), A2(22.09 1.91 years) and A3(31.23 1.99) were investigated. On all three occasions clinical evaluation was conducted. In A2 and A3: total cholesterol, HDL, LDL and triglycerides. In A3 was added to the study of genetic polymorphisms of the ApoE and LDLR. The fragments of interest in this study were amplified by PCR (polymerase chain reaction) and the genotypes were identified by reaction with the restriction enzyme HhaI and HaeIII for ApoE and LDLR polymorphisms, respectively. ApoE genotypes were identified as ε3/ε3 (62.5%), ε3/ε4 (25%), ε2/ε3 (5.4%), ε2/ε4 (5.4%) and ε4/ε4 (1.8%) and the LDLR genotypes identified as AA (85.7%), AT (12.5%) and TT (1.8%). ε2/ε2 genotype was not observed. The analysis of the distribution of ApoE and LDLR genotypes according to the permanence of the dyslipidemia in the study sample showed that all individuals with the ApoE genotype ε2/ε4 and ε4/ε4 kept at least one lipid changes in A2 and A3 and all individuals ApoE genotype ε2/ε3 had not altered lipids in A2 and A3, while for RLDL genotype no difference was found. When analyzed individually, no lipid variable altered in A2 and/or A3, associated with these genotypes, were found. The ApoE gene polymorphism was associated with the permanence of dyslipidemia in young individuals in a longitudinal follow-up study

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Estudos demonstram a associação de alterações da apolipoproteína E (ApoE) e do receptor do LDL (RLDL) com a ocorrência de doenças cardiovasculares e dislipidemia. O objetivo principal deste trabalho foi investigar a associação entre genótipos diferenciais da ApoE e do RLDL com a persistência de alterações de variáveis lipídicas em indivíduos jovens acompanhados há 28 anos no Estudo do Rio de Janeiro (ERJ). Através de um estudo longitudinal, tipo coorte, investigou-se 56 indivíduos (35M) em três avaliações. Em A1 (13.301.53 anos), A2 (22.091.91 anos) e A3 (31.231.99). Nas três ocasiões foi realizada avaliação clínica. Em A2 e A3 foram dosados colesterol total, HDL, LDL e triglicerídeos. Em A3 acrescentou-se o estudo dos polimorfismos genéticos da ApoE e do RLDL. Os fragmentos de interesse neste estudo foram amplificados por PCR (polymerase chain reaction) e os genótipos foram identificados através de reações de restrição. As frequências genotípicas de ApoE foram ε3/ε3 (62,5%), ε3/ε4 (25%), ε2/ε3 (5,4%) ,ε2/ε4 (5,4%) e ε4/ε4 (1,8%) e para os genótipos de RLDL foram AA (85,7%), AT (12,5%) e TT (1,8%). O genótipo ε2/ε2 não foi observado. A análise da distribuição dos genótipos de ApoE segundo a permanência de dislipidemia mostrou que todos os indivíduos com genótipo de ApoE dos tipos ε2/ε4 e ε4/ε4 mantiveram pelo menos um lípide alterado em A2 e A3 entretanto, todos os indivíduos com genótipo de ApoE do tipo ε2/ε3 não apresentaram lípides alterados em A2 e A3. Para o genótipo do RLDL não houve diferença significativa. Quando analisadas isoladamente, não foi identificado nenhum resultado significativo em A2 e/ou A3 associado a estes genótipos. O polimorfismo do gene da ApoE esteve associado à permanência de dislipidemia em indivíduos jovens acompanhados em estudo de seguimento longitudinal / Studies have shown the association of changes in the apolipoprotein E (ApoE) and LDL receptor (LDLR) with the occurrence of cardiovascular diseases and dyslipidemia. The objective of this study was to investigate the association between different ApoE genotypes and LDLR with the persistence of changes in lipid variables in young individuals followed-up 28 years in the study of Rio de Janeiro (ERJ). Through a longitudinal study cohort, 56 subjects (35M) in A1(13.30 1.53 years), A2(22.09 1.91 years) and A3(31.23 1.99) were investigated. On all three occasions clinical evaluation was conducted. In A2 and A3: total cholesterol, HDL, LDL and triglycerides. In A3 was added to the study of genetic polymorphisms of the ApoE and LDLR. The fragments of interest in this study were amplified by PCR (polymerase chain reaction) and the genotypes were identified by reaction with the restriction enzyme HhaI and HaeIII for ApoE and LDLR polymorphisms, respectively. ApoE genotypes were identified as ε3/ε3 (62.5%), ε3/ε4 (25%), ε2/ε3 (5.4%), ε2/ε4 (5.4%) and ε4/ε4 (1.8%) and the LDLR genotypes identified as AA (85.7%), AT (12.5%) and TT (1.8%). ε2/ε2 genotype was not observed. The analysis of the distribution of ApoE and LDLR genotypes according to the permanence of the dyslipidemia in the study sample showed that all individuals with the ApoE genotype ε2/ε4 and ε4/ε4 kept at least one lipid changes in A2 and A3 and all individuals ApoE genotype ε2/ε3 had not altered lipids in A2 and A3, while for RLDL genotype no difference was found. When analyzed individually, no lipid variable altered in A2 and/or A3, associated with these genotypes, were found. The ApoE gene polymorphism was associated with the permanence of dyslipidemia in young individuals in a longitudinal follow-up study