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Otávio T. NóbregaCampus Un iversitário Darcy Ribei roAsa Norte, Brasília – DF, 70910–900 (Brazil)Tel. +55 61 3107 1916, E-Mail nobrega@pq.cnpq.br; otavionobrega@unb.br

Original Research Article

Dement Geriatr Cogn Disord 2012;33:311–317DOI: 10.1159/000339672

Accepted: May 22, 2012 Published online: July 2, 2012

Amerindian Genetic Ancestry Protects against Alzheimer’s Disease

Andrea L. Benedeta Clayton F. Moraesa,b Einstein F. Camargosa,c Larissa F. Oliveirac Vinícius C. Souzad Túlio C. Linse Adriane D. Henriquesc Dayanne G.S. Carmoc Wilcelly Machado-Silvac Carla Nunes Araújoe Cláudio Córdovaf Rinaldo W. Pereirae–g Otávio T. Nóbregaa,d

aPrograma de Pós-Graduação em Ciências Médicas, Universidade de Brasília, bHospital da Universidade Católica de Brasília, cHospital Universitário de Brasília, Universidade de Brasília, dPrograma de Pós-Graduação em Ciências da Saúde, Universidade de Brasília, ePrograma de Pós-Graduação em Patologia Molecular, Universidade de Brasília, fPrograma de Pós Graduação em Educação Física, Universidade Católica de Brasília, and gPrograma de Pós-Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, Brasília, DF, Brazil

Key Words

Alzheimer’s disease � Dementia � Genetic ancestry � Anc estry markers � Continental population � Admixture � Complex disease

Abstract

Background: Alzheimer’s disease (AD) is the mo st com-mo n form of dementia worldwide, and bears remarkable evidence for a differential prevalence among continental populations. In this scenario, estimating ancestry propor-tions in recently admixed populations is a strategy that can help increasing knowledge about the genetic structure of this complex trait. Aim/Methods: Our purpose was to as-sess mean ancestry estimates for the three main parental contributors to the Brazilian contingent (European, African and Amerindian) using a panel of 12 ancestry informative markers. Outpatients with the late-onset form of AD (n = 120) were compared for ancestry levels with non-cognitively impaired subjects (n = 412) in the Midwest Brazil, control-ling for classic clinical, social and anthropometric risk fac-tors. Results: Our findings show a 3-fold greater genetic Amerindian content among control subjects compared to AD patients (p < 0.001). Conclusion: Our results suggest that

the allelic architecture of Native Americans can confer pro-tection against the onset of the disease. Copyright © 2012 S. Karger AG, Basel

Introduction

Alzheimer’s disease (AD) is the most commo n pro-gressive neurodegenerative disorder worldwide, affect-ing nearly 27.7 million people presently and with over 4.5 million new cases diagnosed every year, with major healthcare and socioeconomic impacts [1]. This disease is characterized by memory loss along with injury of any other cognitive function [2, 3] and may exhibit early- or late-onset forms depending on intrinsic pathophysiolog-ical mechanisms [4]. Considered a complex phenotype, late-onset AD’s ethiology largely relies on modifiable factors (e.g. literacy level) and non-modifiable (e.g. ge-netic architecture) [2, 5]. Regardless of the technology employed (from single gene to genome-wide centered), most of the classic AD-related genetic risk factors were identified by the ‘association study’ approach, in which the frequency of a specific genotype is compared among affected and non-affected people. Although useful, this

Dement Geriatr Cogn Disord 2012;33:311–317312 Benedet et al.

approach has methodological biases mostly in stratified population, when spurious associations arise from non-perceived genetic structure within an admixed popula-tion [6].

For traits that bear evidence for differential risk among populations with distinct genetic background (e.g. Europeans and Africans), estimating ancestry propor-tions is a strategy that can help increase knowledge about genetic structure of complex traits [7–10]. Ancestry in-formative markers have been used to ascertain the struc-ture of the main continental populations, thus placing ancestry estimates as the uttermost correction factor in genetic association studies, especially with admixed pop-ulations as in Latin America [6, 10–12]. The Brazilian contingent is highly admixed, bearing a remarkable de-gree of heterogeneity across States and geopolitical re-gions, with roughly 60–75% of its ancestry derived from Iberian whites, 10–30% from West Africans and 5–20% from Native Americans [13, 14]. This scenario makes the Brazilian population suitable for ancestry-related studies, whose results can be useful in approaching efforts to map susceptibility variants for traits differentially distributed with respect to ancestry [15]. So far, few association tri-als performed with the highly admixed Brazilian popula-tion consider genetic ancestry estimates either as main or accessory variables when investigating markers to or the etiology of complex phenotypes.

Studies have linked AD with ethnic differences in sev-eral populations [16, 17], and these reports range from differences in incidence rates to the severity of its mani-festation [16, 18–20]. However, these studies were under the influence of various confounding factors due to cross-cultural, educational and social aspects. Marked differ-ences in health and socioeconomic statuses between racial groups in the USA and Europe, for instance, may hinder biases related to literacy levels and language aspects.

To investigate if the parental contributions to the Brazilian contingent provide grounds to the development of the late-onset form of the AD, we compared ancestry levels of a sample of AD and noncognitively impaired older outpatients, controlling for classic clinical, social and anthropometric risk factors.

Methods

SubjectsThe present study represents an analysis of data from an

ongoing cohort initiative for cognitive assessments in Brasília, Brazil. This city (~2.6 million inhabitants) has the important feature of being planned and constructed to bring the ad-

ministrative capital from the coast to the midwest of Brazil, giving rise to a migration process over the last 50 years. For that reason, the capital’s elderly population (~200,000 inhabit-ants) is considered an expression of the genetic diversity of all Brazilian regions.

Subjects with or without previous diagnosis of AD were consecutively enrolled and clinically followed for a minimum time span of 2 years, either at the Medical Center for the Aged Outpatient at Universidade de Brasília or at the Ambulatory for Elderly Care at Universidade Católica de Brasília. Both in-stitutions are located at the metropolitan area of the Brazilian Federal District and employ skilled staffs for management of cognitive disorders. To participate in this research, each vol-unteer or responsible caregiver provided written consent ap-proved by the Institutional Review Board. Project enrolment eligibility criteria consisted on being aged 60 years or older and to fulfill clinical assessments. Patients who self-declared or whose caregiver informed to bear East-Asian ancestry were not enrolled.

Data CollectionAll clinical assessments were executed by two experienced

geriatricians. Clinical evaluation of each patient consisted of his/her follow-up for a time span of at least 2 years to confirm or rule out the diagnosis of AD or any other form of dementia. All patients entitled as control subjects were followed prospec-tively and rendered as cognitively preserved when no decline was observed during follow-up. Demented individuals could be followed either retrospectively when enrolled with prior di-agnosis of AD or by prospective means whenever newly admit-ted, newly diagnosed amongst the nondemented or followed at the centers for less than 2 years before the beginning of the study. The diagnosis or confirmation of probable AD was made according to NINCDS/ADRDA criteria and determined by the clinicians on the basis of consultation with the patient, interview with a knowledgeable informant, review of medical records, neuropsychological assessments and/or laboratory tests whenever applicable.

At admission, all patients were assessed with a validat-ed Brazilian Portuguese version of the Mini-Mental State Examination (MMSE), whereas the Clinical Dementia Rating (CDR) scale was obtained for demented patients only. Also at admission, data as age (years), waist circumference (WC; cm), systolic (SBP; mm Hg) and diastolic (DBP; mm Hg) blood pressure and literacy level were collected. This latter variable was assessed in terms of having either a complete basic cycle of educational or higher (≥4 years) or an incomplete basic cycle or no formal education at all (<4 years). For patients enrolled with prior diagnosis of AD or throughout our assessments, baseline characteristics consisted of data collected at the year of onset of the disease.

Markers Selection and GenotypingFor laboratory experiments, peripheral venous blood

samples were collected into EDTA-containing tube and DNA extraction was performed using standard extraction kits (QIAamp DNA Mini Kit, Qiagen, Brazil). For individual ge-netic ancestry estimates, 12 nonlinked, autosomal ancestry in-

Dement Geriatr Cogn Disord 2012;33:311–317 313Genetic Ancestry and Alzheimer’s Disease

formative markers (AIMs) that are highly informative due to very different allele frequencies (δ > 0.6) among parental pop-ulations were selected based on the assumption that few, well-defined AIMs are as good as a large number of random SNPs to indicate substructure. From those, eight displayed a large difference between European and Native American frequen-cies (rs730570, rs3796384, rs1426654, rs734780, rs1129038, rs2278354, rs2065160, rs4305737), five between European and African frequencies (rs803733, rs1426654, rs2814778, rs1129038, rs1240709), and two between African and Native American frequencies (rs2814778, rs3138521). Allelic fre-quencies from the parental populations were retrieved from mapping studies of unrelated ethnic samples [21, 22], and con-firmed using a public genomic database [23].

Since eleven of the selected AIMs were single nucleotide polymorphisms (SNPs), genotyping was conducted by a single base extension procedure followed by capillary electrophore-sis. Briefly, DNA fragments encompassing the polymorphic site and sized 100–200 pb were amplified by standard poly-merase chain reaction (PCR) using the Qiagen Multiplex PCR Kit. Multiplex reactions were optimized with primers seg-regated into two sets (set 1: rs730570; rs3796384; rs803733; rs1426654; rs734780 / set 2: rs2814778; rs1129038; rs1240709; rs2278354; rs2065160; rs4305737) according to the size of the single-base sequencing primer, and co-amplified in a Veriti™ Dx Thermal Cycler (Applied Biosystems) using the following cycles: denaturation at 95°C for 15 min, followed by 39 cycles of 30 s at 94°C, 90 s at 57°C and 60 s at 72°C, with a final exten-sion step at 72°C for 10 min. Following amplification, products were purified to eliminate nonincorporated dNTPs and prim-ers by adding 1 U of Exo I and 1 U of CIAP to 3 μl of each PCR product corrected to have 1X of the CIAP reaction buffer. This mixture was incubated for 60 min at 37°C followed by denatur-ation at 75°C for 15 min.

For minissequencing, each multiplex was genotyped by single-base extension reaction using the SNaPshot Multiplex System kit (Applied Biosystems, USA) following manufactur-er’s recommendations. A second purification step was carried out by mixing 1 U of CIAP with 5 μl of the single-base exten-sion reaction corrected to 1X CIAP reaction buffer, which was incubated for 1 min at 37°C and for 15 s at 85°C. Following addition of 1 μl of each purified product to 8.83 μl Hi-Di for-mamide and 0.17 μl GS120 Liz internal size standard, capillary electrophoresis was carried on an ABI Prism 3130XL genetic analyzer (Applied Biosystems) using ABI 3700 POP 7 polymer. The obtained data were analyzed in the GeneMapper software (Applied Biosystems). Eletropherograms were rendered suc-cessful when only mild noise was observed and allowed coinci-dent identification of the polymorphic site by two independent readers after visual inspection. Samples with less than 90% call rate were repeated, after which blanks were filled with uni-tary (singleplex) reactions until the entire panel of SNPs was fulfilled.

To genotype the Indel marker (rs3138521), standard PCR was performed in 10 μl in conditions as follows: with 1X Taq polymerase buffer, 0.25 mm dNTP, 0.16 mg/μl BSA, 1.5 μm MgCl2, 1 U Taq polymerase and 10–40 ng of DNA. For ther-

mocycling, samples were submitted to 95°C for 5 min and then to 29 cycles of 1 min at 95°C, 1 min at 54°C and 1 min at 72°C, with final extension for 20 min at 72°C. Genotypes were as-sessed directly after a 2% agarose electrophoresis gel. To gen-otype the Apolipoprotein E gene (Apo E) and determine the classic e2, e3 and e4 alleles for statistic control, a PCR method based on a multiplex refractory mutation system was adapted from Donohoe et al. [24], followed by electrophoresis in a 1.6% agarose gel. Quality control for the AT3 marker consisted of randomly selecting 50 samples to be re-genopyped whereas for the ApoE polymorphism consisted of systematic repetition of all e2 and e4 carriers. All reassessments were done by an in-dependent technician, and genotyping success rate and con-cordance scored at 100%. Concentration and sequence of all primers used are available upon request.

Statistical AnalysisIndividual genetic ancestry was estimated with an algo-

rithm based on maximum likelihood estimation (MLE) [25]. Briefly, the log likelihood function is maximized for the ad-mixture parameter of up to 3 parental populations using their a priori known allele frequencies as well as the corresponding frequencies in the admixed samples. The MLE approach was implemented in the software program IAE3CI, and detailed statistics are available elsewhere [26, 27].

Cases were labeled as positive (affected) or negative (nonaf-fected) regarding clinical diagnose of AD. The Kolmogorov-Smirnov test was performed to check for normal distribution of the variables within each group. The Student’s t test and the χ2 test were performed for comparison of classic clinical, social and anthropometric data across subjects affected and nonaf-fected by AD. For the nonnormally distributed ancestry scores, the Mann-Whitney test was used to compare these traits be-tween affected and nonaffected. With the aim of achieving a close to normal distribution to allow an analysis of covariance (ANCOVA) using age, gender, waist circumference, systolic and diastolic blood pressure, literacy level and presence of the Apo e4 allele as covariables, the data on ancestry estimates were log-transformed (log10).

For all tests, p < 0.05 was considered statistically significant. The software SPSS, version 19 (SPSS Inc., Chicago, Ill., USA) was used for statistical calculations.

Results

From 2009 through 2011, data from 630 participants (~0.32% of the whole elderly segment in Brasília; aged from 60 to 96 years) were gathered according to the in-clusion criteria. The number of clinical interviews with each patient ranged from 2 to 11 different consultations throughout up to 9 years of follow-up, with the group of AD patients displaying significantly more encounters with the clinical staff (5.8 ± 2.7 consultations) and more years of follow-up (5.2 ± 3.4 years) compared to the non-

Dement Geriatr Cogn Disord 2012;33:311–317314 Benedet et al.

demented group (3.3 ± 1.2 consultations and 2.5 ± 0.5 years, respectively).

On a whole-group analysis, our results show a remark-able degree of population structure given the spectrum of intermediate frequencies of all AIMs in our sample compared to parental populations (table 1). In addition, 2 AIMs deviated from the Hardy-Weinberg equilibrium (rs1129038 e rs1426654), corroborating the recent ad-mixture of this city. Our panel of 12 autosomal markers assures the trihybrid structure of our sample and reveals average genetic contributions (56.8% Europeans, 29.3% Africans, and 13.9% Native Americans) comparable to ancestry levels found elsewhere in Brazil [14] and Latin America [28].

From all participants, 120 individuals were diagnosed with AD whereas 412 subjects were identified as nonde-mented, cognitively healthy subjects. The remaining 98 ex-hibited other cognitive disorders not compatible with AD, including cases of mixed dementia, and were ruled out. To assess homogeneity among the 532 eligible for analysis, classic clinical, social and anthropometric data considered at baseline were compared between subjects affected and nonaffected by AD (table 2). Literacy level as well as waist circumference, age and systolic blood pressure means were considered equal in both groups. Nonetheless, dif-ferences in gender and DBP were significant, with the control group being higher in diastolic pressure and over-represented with women. As expected, carriers of the e4

allele of Apo E were more frequent in AD patients (47.5%) than in cognitively normal subjects (19.7%).

To evaluate ancestry proportions among nonaffected and AD patients, the non-parametric Mann-Whitney test was performed and significant differences were ob-served for all estimates of genetic ancestry analyzed. European and African ancestry proportions were signifi-cantly higher whilst the Amerindian ancestry proportion was lower for AD patients (fig. 1). An analysis of co-vari-ance using log-transformed ancestry estimates showed that the significance of these differences resisted adjust-ment to important contributing factors to the late-onset form of AD, with emphasis to age, gender and literacy level with the most prominent influence on the model. Mean ancestry estimates did not differ across different clusters of Apo E genotypes tested (data not shown), with or without adjustment.

Discussion

The present study shows significant differences in genetic ancestry proportions between AD patients and noncognitively impaired subjects, with AD patients hav-ing an average content of Amerindian genetic ancestry threefold lower (5.6 vs. 16.2%; p < 0.001) than the cor-

Table 1. Description of the ancestry informative markers, their alleles, chromosome positions and frequencies in the European (EUR), African (AFR) and Amerindian (AMR) parental populations

Locus Position Alleles Frequencies

sa mple EUR AFR AMR

rs730570 14q32 A 0.676 0.860 0.185 0.100rs3796384 3p14 C 0.384 0.154 0.783 0.875rs803733 9q33 C 0.621 0.880 0.015 0.411rs1426654 15q21 C 0.261 0.000 0.980 0.950rs734780 15q26 C 0.322 0.070 0.710 0.854rs2814778 1q23 A 0.221 0.998 0.001 1.000rs1129038 15q13 C 0.625 0.224 0.995 0.983rs1240709 1p36.3 A 0.522 0.794 0.036 0.103rs2278354 5p15.2 G 0.282 0.120 0.704 0.839rs2065160 1q32 C 0.241 0,078 0.512 0.850rs4305737 6q24 A 0.431 0.250 0.929 1.000rs3138521 1q25 Insertion 0.369 0.282 0.858 0.061

Source: Shriver et al. [21]; Smith et al. [22]; HapMap [23].

Table 2. Comparison of continuous and categorical variables across AD patients and control, nondemented subjects at baseline

Variable AD(n = 120)

Control(n= 412)

p*

mean SD me an SD

Age, years 072.3 07.8 070.8 08.2 <0.38WC, cm 093.5 10.4 092.1 10.0 <0.53SBP, mm Hg 134.1 20.3 137.5 22.0 <0.80DBP, mm Hg 079.3 10.6 082.1 13.1 <0.01MMSE (points) 013.7 07.1 024.5 03.3 <0.001

n % n % p+

Male 41 34.2 48 11.6 <0.001e4 carriers 57 47.5 81 19.7 <0.001<4 years schooling 73 60.8 231 56.1 <0.68CDR ≤ 2 52 43.3 0– 0– 0–

n stands for absolute number and % denotes proportion.* Significance verified by Student’s t test. + Significance verified

by the χ2 test.

Dement Geriatr Cogn Disord 2012;33:311–317 315Genetic Ancestry and Alzheimer’s Disease

responding, average content found in cognitively nor-mal older adults. Our panel of 12 AIMs indicates that Amerindian allelic architecture could confer protection against the development of AD. Little is known about AD in the American Indian populations, and there are no prior results in the South American scenario that can support this assumption, but a study by Hendrie et al. [29] reported a considerably lower age-adjusted preva-lence of AD among Cree Native Americans compared to subjects without that heritage. Also, Rosenberg et al. [30] found that persons with a greater degree of Cherokee Indian heritage had a lower risk of developing AD.

Until now, one single report sought to investigate the relationship between genomic ancestry and the AD phe-notype in Brazil [31]. According to their report, African ancestry protects against AD-related neuropathology. Nonetheless, their analysis was designed to differentiate European-only ancestry from admixed individuals (in-cluding heritage from Native Americans), which could have elapsed unnoticed the Amerindian contribution to the protection effect.

In our conditions, differences in gender distribution between affected and non-affected individuals probably derived from cultural aspects, since older women tend to be greater consumers of outpatient services, and older men of inpatient healthcare [32]. Despite these discrep-ancies render important confounding factor, age and gender were controlled in our analysis of covariance. Lower DBP is attributable to the frequent dysautonomia of AD patients.

Regarding social profile, the prevalence of individu-als with less than 4 years of formal education is consis-

tent with the general low literacy level of older adults in Brazil, which, in turn, is more correlated with low in-come and poor healthcare countrywide than self-rated ethnical classification [33–35]. This rather homoge-neous literacy profile shown for AD and control subjects at baseline tends to attenuate disparities related to socio-economic status. Despite this study tends to pose a con-tribution by controlling influences, there are important limitations to acknowledge. Remarkable heterogene-ity produced by differences in pharmacotherapy (prior or current) and by the sampling method not based on the pattern of clinical evolution, among other factors not considered herein, may add complexity to this sce-nario. Moreover, the statistical power of the 12 AIM panel might have been insufficient to accurately precise ancestry in the admixed population according to what is advised elsewhere [11], and this might prompt some caution in the interpretation of our results. However, when our ancestry estimates are compared with those from different Brazilian samples estimated with a higher number of markers, proportions are quite similar, espe-cially with regard to levels of Amerindian ancestry [36]. Another bias akin to almost all Brazilian-based genetic ancestry studies concerns the Amerindian informative markers used since they are usually based on the na-tive North American population which, depending on the panel of markers, might display considerable dif-ference when compared to South Native Americans. In this matter, we recall that the AIMs used herein display remarkable allelic frequency differences, and suitable to distinguish between the major continental populations considered.

F i g. 1. Comparison of mean estimates of European (a), African (b) and Amerindian (c) genetic ancestries across individuals affected and nonaffected by AD. Significance was verified by the Mann-Whitney test without adjustments. Vertical bars represent intervals of 2 SEs.

64

62

60

58

54

52AD Control

p = 0.001

56Eu

rope

an a

nces

try

(%)

AD Control

p = 0.00538

36

34

32

28

26

30

Afr

ican

anc

estr

y (%

)

AD Control

20

17

14

11

5

2

8

p < 0.001

Am

erin

dian

anc

estr

y (%

)

a b c

Dement Geriatr Cogn Disord 2012;33:311–317316 Benedet et al.

In summary, our findings suggest that Amerindians could have a protective genetic configuration that can help avoiding the development of the late-onset AD. Mapping by admixture linkage disequilibrium, for in-stance, may be necessary to confirm this hypothesis and to refine the search for elements in the Amerindian ge-netic architecture that might protection against or pre-dispose to this form of dementia.

Acknowledgments

This work was supported by FAPDF (grant # 193.000.449–2008), Finatec/UnB (grant # 5563/2009) and DPP/UnB (UnBDoc 121696/2011 and 65226/2012) to O.T. Nóbrega. Dr. R.W. Pereira was supported by a fellowship for productivity in research from CNPq.

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