Revista Clínica de la Escuela de Medicina UCR – HSJD Año 2014 Vol 4 No VI Rev Cl EMed UCR www.revistaclinicahsjd.ucr.ac.cr 30 Setiembre 2014 25 ARTÍCULO ORIGINAL: Determination of ApoE gene in patients with mild cognitive impairment Hospital San Juan de Dios, San José, Costa Rica. Fundado en 1845 First report in Costa Rica Recibido: 26/03/2014 Aceptado: 21/04/2014 Norbel Román Garita1 Carolina Boza Calvo2,5 Leonardo Calvo Flores3,5 Alia Kozakova Valchuk4,5 Adriana Von Storren Cortés4 Luis Sequeira Rojas4 1Memory and Aging Clinic. Neurology Department. Hospital San Juan de Dios. E-mail: drnorbelro- man@racsa.co.cr 2Centro de Investigación en Hematología y Trastornos Afines. Universidad de Costa Rica. Memory and Aging Clinic. Hospital San Juan de Dios. E-mail: cbozac@gmail.com 3Centro de Investigación en Hematología y Trastornos Afines, Universidad de Costa Rica. Hospital San Juan de Dios. 4Geriatrics and Gerontology Department. Hospital San Juan de Dios. 5Medical School, University of Costa Rica. RESUMEN Introducción: La enfermedad de Alzheimer (EA) es la causa más común de demencia. Es una enfermedad multifactorial en la que las condicio- nes genéticas y ambientales interactúan para presentar una manifestación clínica. El genotipo e4 de la apolipoproteína E (ApoE4) constituye un factor de riesgo para el desarrollo de la EA. Estimaciones indican que el alelo ApoE4 se presenta en el 15-16% de la población en gene- ral, con una mayor presencia en poblaciones caucásicas y casi en el 50% de los pacientes con EA. La presencia del genotipo de ApoE4 aumen- ta el riesgo de desarrollar EA de 3 a 8 veces más y disminuye la edad de aparición de la enferme- dad entre 7 a 15 años antes. En forma homocigo- ta el riesgo aumenta 33 veces. En pacientes de aparición tardía se encuentra en el 65% de los casos y el porcentaje se eleva al 80% cuando hay presencia de un familiar con EA. ApoE sigue siendo el biomarcador por excelencia para la predicción y el diagnóstico de EA. Objetivo: Estandarizar la técnica y determinar la frecuencia de ApoE en los 4 alelos de importancia clínica en los pacientes con deterioro cognitivo leve. Méto- dos: Se seleccionó una muestra de 14 pacientes previamente evaluados en la Clínica de Memoria y Envejecimiento del HSJD, diagnosticados con deterioro cognitivo leve amnésico. Se recogieron ISSN 2215-2741 Revista Clínica de la Escuela de Medicina UCR – HSJD Año 2014 Vol 4 No VI Rev Cl EMed UCR www.revistaclinicahsjd.ucr.ac.cr 30 Setiembre 2014 26 muestras de sangre y se realizó el protocolo de extracción de ADN de Miller et al. Posterior- mente se realizó una PCR múltiple análisis si- multáneo de los alelos de ApoE. Resultados: Se están analizando un total de 14 pacientes selec- cionados según los criterios definidos por el equipo interdisciplinario, con el fin de determi- nar la presencia de los alelos de APOE en esta población. Conclusiones: La frecuencia de la presencia del gen ApoE permite describir las características de la población de Costa Rica como un factor de riesgo para el desarrollo de AD. PALABRAS CLAVE Apoliproteína E. ApoE4. Enfermedad Alzhei- mer. Deterioro cognitivo leve. Diagnóstico tem- prano. Biomarcador ABSTRACT Background: Alzheimer's disease (AD) is the most common cause of dementia. It is a multifac- torial disease in which genetic and environmental conditions interact to present a clinical manifes- tation. The e4 genotype for the apolipoprotein E (ApoE4) is a risk factor for developing AD. ApoE4 presents 15-16% of the general popula- tion, with greater presence in Caucasian popula- tions and nearly in 50% of subjects with AD. The presence of ApoE4 genotype increases the risk of developing AD 3 to 8 times higher and decreases age onset between 7 to 15 years. In homozygous form the risk increases 33 times. In late-onset AD is found in 65% of the cases and the percentage rises to 80% in presence of a family member with EA. ApoE remains the biomarker for predicting and diagnosing AD. Objective: To standardize the technique and determine the frequency of the ApoE’s 4 alleles of clinical significance in patients with mild cognitive impairment. Methods: Patients who were previously evaluated in the Memory Clinic- Hospital San Juan de Dios and diagnosed with mild cognitive impairment were selected. We collected blood samples and performed DNA extraction protocol by Miller et al. Multiplex PCR was performed in 14 patients for the simul- taneous analysis of gene ApoE genotype of the samples. Results: We are testing a total of 14 patients diagnosed with mild cognitive impair- ment who were previously diagnosed by the interdisciplinary team to determine the presence of the ApoE gene. Conclusions: The frequency of the presence of the ApoE gene allows to de- scribe the characteristics of the Costa Rican population as a risk factor for developing AD KEY WORDS Apoliprotein E. ApoE4. Alzheimer’s disease, Mild Cognitive Impairment. Early diagnosis. BACKGROUND Alzheimer's disease (AD) is the most common cause of dementia. It is a multifactorial disease in which genetic and environmental conditions interact to present a clinical manifestation(1). The e4 genotype for the apolipoprotein E (ApoE4) is a risk factor for developing AD. ApoE4 presents 15-16% of the general population(2), with greater presence in Caucasian populations(3) and nearly in 50% of subjects with AD. The presence of ApoE4 genotype increases the risk of developing AD 3 to 8 times higher and decreases age onset between 7 to 15 years(4). In homozygous form the risk increases 33 times(2,5). In late-onset AD is found in 65% of the cases(6,7) and the percent- age rises to 80% in presence of a family member with EA(8). ApoE remains the biomarker for predicting and diagnosing AD(10). Figure 1. Structural protein conformation of the E4 and E3 haplotypes of ApoE Source: http://biomodel.uah.es/model2/lip/apo-e4.htm In Costa Rica, the first prevalence study con- ducted in a community (Santo Domingo de Heredia) with a sample of 400 subjects, showed a 4.2% prevalence of probable dementia (in any of its subtypes)(12). Among the subjects evaluated 41 were diagnosed with AD (n = 14) and mild cognitive impairment (MCI, n = 27). Revista Clínica de la Escuela de Medicina UCR – HSJD Año 2014 Vol 4 No VI Rev Cl EMed UCR www.revistaclinicahsjd.ucr.ac.cr 30 Setiembre 2014 27 At the Memory and Aging Clinic of the Hospital San Juan de Dios (CMEC) interdisciplinary diagnosis by consensus assessments have been conducted for the past 7 years, using a protocol established by the our team of neurologists, geri- atricians and clinical psychologists, which in- cludes a battery of tests for assessing cognitive and functional performance (screening, medical history, neuropsychological assessment, neuro- logical examination, review of the patients rec- ord, molecular biology studies and neuroimag- ing). In 2012 the CMEC reports a first analysis of the prevalence of MCI an AD in the population served by the clinic (n = 128), during 2010-2011. The most frequent diagnosis was MCI (44,5%), while dementia were found in 30.5% of cases, where the AD (43,6%) and vascular dementia (25,6%) predominated. Graphic 1. Final diagnosis Source: Memory and Aging Clinic, HJSD. These results showed that the CMEC is attracting patients at early stages, so our efforts should focus on this population. This highlights the importance of providing follow-up to patients and also reinforces the need to implement the detection of biomarkers and the presence of other genetic mutations considered as risk factors for dementia (ApoE), as a part of the diagnostic process. Thus, during 2012 there was a national campaign for early diagnosis, promoted by the Costarrican Alzheimer and Other Dementias Association (ASCADA), which gave the first donation of the ApoE detection kits. The aim of this study is to standardize the tech- nique for detection of the ApoE haplotypes in DNA samples, and determine the frequency of the ApoE 4 alleles of clinical significance in patients with Amnesic Mild Cognitive Impair- ment (MCI-A), evaluated and diagnosed by the CMEC. METHODS AND MATERIALS Patients were previously evaluated in the Memory Clinic-Hospital San Juan de Dios using a protocol established by our team of neurolo- gists, geriatricians and clinical psychologists, which includes a battery of tests for assessing cognitive and functional performance (screening, medical history, neuropsychological assessment, neurological examination, review of the patient’s record, molecular biology studies and neuroim- aging). Subjects gave written informed consent and the University of Costa Rica’s institutional Bioethics review board approved the study. Pa- tients diagnosed with MCI-A were randomly selected. We collected blood samples and performed DNA extraction protocol by Miller et al, 1988(11). Mul- tiplex PCR was performed in 14 anonymous samples for the simultaneous analysis of gene ApoE genotype of the samples. PCR product ApoE was then analyzed by electrophoresis at 105 V for 35 min through a 2% agarose gel and stained with ethidium bromide. RESULTS We are testing a total of 14 anonymous samples of individuals diagnosed with MCI-A who were previously diagnosed by the interdisciplinary team to determine the presence of the ApoE gene. They were divided by according to amnesic MCI’s subtypes (simple or multodomain). Table 1. Distribution of Mild Cognitive Impairment (MCI) in patients previously evaluated in the Memory Clinic-Hospital San Juan de Dios (n = 14). MCI* Frequency MCI-Am 71,43 MCI-A 28,57 (*)MCI-Am: Amnestic multidomain MCI, MCI-A: Amnestic MCI. MCI   Dementia   Other  diagnosis   N/A   None  %   44%   31%   17%   5%   3%   0% 20% 40% 60% Final diagnosis 2010-2011 n=128 Revista Clínica de la Escuela de Medicina UCR – HSJD Año 2014 Vol 4 No VI Rev Cl EMed UCR www.revistaclinicahsjd.ucr.ac.cr 30 Setiembre 2014 28 All of the samples were processed, but only fourth were able to determine their ApoE geno- type. This due to a complex process of standardi- zation that took longer than initially expected. The following are the preliminary results of the samples analyzed. Table 2. Distribution of ApoE genotypes in patients previously evaluated in the Memory Clinic-Hospital San Juan de Dios (n = 14). Genotype/Haplotypes Frequency In process1 71,43 E3/E4 14,29 E3/E3 14,29 112cys2 92,86 Other 7,14 1) Samples still not totally processed. 2) Protein conformation associated with E2 and E3 haplo- types. Figure 2. Multiplex PCR amplification of ApoE gene DISCUSSION AND CONCLUSIONS This is the first study of standardization of the test for ApoE genotype in Costa Rica. Despite the constraints faced, we now have solved the problems with the standardization of the tech- nique and have the capacity to continue the anal- ysis of the patients. We overcome the most complicated stage for the application, which constituted the adaptation of the technique to our environment. This is a breakthrough because it is the stage that more time and resources involve. Already achieved preliminary results of 4 sam- ples of a new technique in the country, represent- ing a pioneering project in our field, allowing it to provide the first results which will be statisti- cally significant once more samples are pro- cessed. ACKNOWLEDGMENTS This article was possible by the support of Vicerrectoría de Investigación, Universidad de Costa Rica (project No. 807-B2-283) and Es- cuela de Medicina, Universidad de Costa Rica (project No. 422-B2-324). REFERENCIAS BIBLIOGRÁFICAS 1. Román N Boza C. Revisión sobre la re- lación del genotipo para ApoE4 y el desarrollo de demencia tipo Alzheimer. Rev Cl EMed UCR. 2012;2(5). ISSN 2215-2741. 2. Salvia N Clarimon J. Genética en la Enfermedad de Alzheimer. Rev Neurol 2010;50(6):360- 364. 3. Crean S Ward A Mercald C et al. Apolipoprotein E 4: Prevalence in Alz- heimer’s disease patients varies across global population: A Systematic Litera- ture Review and MetaAnalysis. Dement Geriatr Cogn Disord 2011;31:20-30. 4. 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