APOE Variants in an Iberian Alzheimer Cohort Detected through an Optimized Sanger Sequencing Protocol

Ricardo D González, Iva Gomes, Catarina Gomes, Rita Rocha, Luís Durães, Patrícia Sousa, Manuel Figueruelo, Maria Rodríguez, Carmen Pita, Roberto Hornero, Carlos Gómez, Alexandra M Lopes, Nádia Pinto, Sandra Martins, Ricardo D González, Iva Gomes, Catarina Gomes, Rita Rocha, Luís Durães, Patrícia Sousa, Manuel Figueruelo, Maria Rodríguez, Carmen Pita, Roberto Hornero, Carlos Gómez, Alexandra M Lopes, Nádia Pinto, Sandra Martins

Abstract

The primary genetic risk factor for late onset Alzheimer's disease (LOAD) is the APOE4 allele of Apolipoprotein E (APOE) gene. The three most common variants of APOE are determined by single nucleotide polymorphisms (SNPs) rs429358 and rs7412. Our aim was to estimate allele and genotype frequencies of APOE variants in an Iberian cohort, thus helping to understand differences in APOE-related LOAD risk observed across populations. We analyzed saliva or buccal swab samples from 229 LOAD patients and 89 healthy elderly controls (≥68 years old) from Northern Portugal and Castile and León region, Spain. The genotyping was performed by Sanger sequencing, optimized to overcome GC content drawbacks. Results obtained in our Iberian LOAD and control cohorts are in line with previous large meta-analyses on APOE frequencies in Caucasian populations; however, we found differences in allele frequencies between our Portuguese and Spanish subgroups of AD patients. Moreover, when comparing studies from Iberian and other Caucasian cohorts, differences in APOE2 and APOE4 frequencies and subsequent different APOE-related LOAD risks must be clarified. These results show the importance of studying genetic variation at the APOE gene in different populations (including analyses at a regional level) to increase our knowledge about its clinical significance.

Keywords: Apolipoprotein E (APOE); disease risk; late onset Alzheimer’s disease (LOAD); rs429358; rs7412; sanger sequencing.

Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Sequenced region for Apolipoprotein E (APOE) including single nucleotide polymorphisms (SNPs) rs429358 (squared T in figure) and rs7412 (squared C in figure). Sequence downloaded from UCSC Genome Browser (https://genome.ucsc.edu) Assembly Dec. 2013, GRCh38/hg38, Chr19: 44908567-44909018. Designed forward and reverse primers for amplification and sequencing of target region are represented in italic and bold.
Figure 2
Figure 2
Schematic representation of the optimized protocol for Apolipoprotein E (APOE) genotyping.

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