Apolipoprotein E polymorphism and functional disability in Brazilian elders : the Bambuí Health and Aging Study

Numerous studies have associated the apolipoprotein E (apoE) ε4 allele with worse health status, but few have assessed the existence of genotypedependent variations in functional performance. Among participants in the Bambuí Health and Aging Study, Minas Gerais State, Brazil, 1,408 elderly underwent apoE genotyping. Functionality was assessed with a questionnaire, and individuals were classified as dependent in basic activities of daily living (BADLs), instrumental activities of daily living (IADLs), and mobility. The association between apoE genotype and functional status was assessed by logistic regression, taking confounding factors into account. Presence of ε4 allele was associated with lower odds of mobility deficit (OR = 0.65; 95%CI: 0.47-0.92) in the adjusted analysis. There were no significant differences in relation to presence of dependency in BADLs and IADLs. The reasons are not entirely understood, but they may involve the role of ε4 allele as a “thrifty gene” in a sample exposed to high risk of infectious and nutritional diseases in the past. Apolipoprotein E4; Disabled Persons; Aged http://dx.doi.org/10.1590/0102-311X00080115 Correspondence


Introduction
The apolipoprotein E (apoE) gene has been extensively studied as an aging-related gene.Beyond its role in lipid metabolism, apoE is associated with atherosclerosis, neuronal repair and maintenance, inflammation and, possibly, immune response to infectious diseases 1 .Several studies have shown an association between the ε4 allele (rs429358) and age-related diseases such as coronary heart disease 2,3,4 , stroke 5 and Alzheimer's disease 6,7 .The effect of this genotype in mortality has also been widely investigated, but the results are not consistent across all populations 8,9,10,11 .
Researches have suggested that the apoE gene should be considered a "frailty gene" that increases the age-specific susceptibility to death 12 .If the apoE gene really is a "frailty gene", it would be reasonable to expect ε4 carriers to accumulate more age-related diseases and to demonstrate poor functional performance in advanced ages 13,14 .A small number of studies have addressed genotype-dependent variations on functional performance of the elderly and have reported conflicting results.In some populations, no significant association between the apoE genotype and functional decline has been detected 15,16 , while in other cases, the ε4 allele was associated with worse functional status among men only 17 or among women only 18 .Two previous studies related the presence of ε4 allele to poor functional performance 19,20 , but the same allele was considered a protection factor against activities of daily living impairments among elderly Americans 14 .Impairments in instrumental activities of daily living functional performance were more prevailent in elderly men carrying ε2 allele (rs7412) in just one sample 21 .
Although different studies present a variation in the approach to assessing functional status, the results have shown a strong discordance with regard to the association between functional status and the apoE genotype among the aged.It is also important to point out that most of these studies were conducted in Europe and North America, with just one study conducted in Taiwan 15 .
The present study aims to evaluate the association between the apoE genotype and functional status, considering performance in instrumental activities of daily living (IADL), basic activities of daily living (BADL) and mobility-related tasks, in an aged and low-educated population in Brazil.

Sampling study
The Bambuí Health and Aging Study has been carried out since 1997 in Bambuí, a city of 15,000 inhabitants located in Minas Gerais State (Southeastern Brazil).The leading causes of death among elderly residents in Bambuí are stroke, Chagas disease and coronary heart disease.Bambuí has been an endemic area for Chagas disease -a parasitic disease caused by the protozoan parasite Trypanosoma cruzi, and the rate of T. cruzi infection is still high among elderly residents due to a cohort effect 22,23 .
The cohort baseline survey was conducted in 1997 and the study population was identified by full census report in the city.All residents aged 60 or older (1,742) were invited to join the study; 1,606 were interviewed (92.2%) and 1,496 (85.9%) were submitted to physical examinations and had blood samples collected.Details on the methodology and participants' health profile in the baseline are available in previous papers 22,23 .
The original study proposal of the Bambuí Health and Aging Study was approved by the Ethics Research Committee at Oswaldo Cruz Foundation (Fiocruz) in Rio de Janeiro with an informed consent form signed by the participants at baseline and at each subsequent visit.

Functional measurements
Functional disability was assessed regarding selfreported performance on basic activities of daily living (BADL), instrumental activities of daily living (IADL), and mobility-related tasks 24,25,26 .The participants were asked whether they experienced any difficulties in any of the following BADL: bathing, dressing, getting in and out of bed and feeding.Questions about being able to manage money, do household chores and prepare food on their own were also asked in order to assess IADL performance.The mobility-related questions concerned any difficulties in walking 100 yards, climbing stairs, stooping, squatting and kneeling, lifting and carrying weights, walking across a room, getting up from a chair and walking without assistance.The candidates who reported being unable to perform at least one of the tasks without help for each domain were considered disable in BADL, IADL or mobility.

DNA extraction, PCR amplification and RFLP genotyping
Genomic DNA was obtained from the blood samples using the Wizard genomic DNA extraction kit (Promega, Madison, USA) and were subjected to the polymerase chain reaction amplification, using the following primers: forward 5' TAA GCT TGG CAC GGC TGT CCA AGG A 3' and reverse 5' ACA GAA TTC GCC CCG GCC TGG TAC AC 3'.The amplified DNA was subjected to the restriction fragment length polymorphism (RFLP) technique, with digestion by HhaI, reproducing the following patterns: ε2ε2, 83 and 91 bp; ε3ε3, 91, 48, and 35 bp, and ε4ε4, 72, 48, 35, and 19 bp 27 .These fragments were visualized on 4% agarose gels.

Confounders
Sociodemographic factors such as age, gender, skin color and schooling had been considered potential confounding factors and were used as adjustment variables, as well as smoking status, mini-mental scores, hypertension, diabetes, coronary heart disease, stroke, LDL and HDL cholesterol levels and C-reactive protein (CRP) levels.
Schooling was assessed by numbers of completed years of education (0 to 3 years and 4 years or more).Skin color grading was based on the different skin color that individuals displayed in photographs.This variable was dichotomized into white and non-white for the study analysis.Individuals who had smoked at least 100 cigarettes in their lifetime and were still smoking were considered current smokers.
Cognitive functioning was assessed using a Portuguese version of the Mini-Mental State Examination (MMSE).The cut-off used in this population was 22, as defined in an earlier publication 28 .Stroke 29 and coronary heart disease 30 were assessed by means of standardized questionnaires.
Standard desk mercurial sphygmomanometers (Tyco's 5097-30, USA) and stethoscopes (Littman's Cardiology II, USA) were used for measuring blood pressure.Blood pressure was measured 30 or more minutes after the last caffeine intake or cigarette smoked.Three measures were taken after 5 minutes of initial rest and subsequently at 2-minute intervals.Hypertension was defined by mean systolic blood pressure > 140mmHg and/ or mean diastolic blood pressure > 90mmHg and/or current use of hypertension medication.
Biochemical analyses (fasting glucose, HDL cholesterol, LDL cholesterol and triglyceride) were determined with standardized enzymatic methods, using automated equipment (Eclipse Vitalab; Merck, Netherlands).Diabetes mellitus was defined as a fasting blood glucose level equal to or higher than 126mg/dL and/or current use of hypoglycemic medication.Highly sensitive Creactive protein levels were determined using the immunonephelometric method (High Sensitive CRP, Dade Behring, Germany).
All interviews were conducted in the participants' homes by trained and certified interviewers, and were answered by the participant or, if they suffered from a life-limiting condition, by a close relative.Blood collection and examinations were performed at the clinic.More details can be found in previous publications 23 .

Data analysis
Descriptive analyses of all variables were performed using absolute frequency, relative frequency, mean, standard-deviation, median and 25th and 75th percentiles for the total population and for each genotypic group.
Association between self-reported functional disability and the apoE genotypes (or the presence of an apoE ε4 allele) were measured using logistic regression.Odds ratio (OR) and 95% confidence intervals (95%CI) were calculated for each domain (BADL, IADL and mobility).The analysis was adjusted for potential confounding factors.The Hosmer-Lemeshow test and residual plots were used to evaluate if the model was suitable.
The statistical analysis was performed by means of version 13.0 of the Stata software (Stata-Corp LP, College Station, USA) and a significance level of 0.05 was considered.
Table 1 shows the baseline characteristics of the study population according to the genotype groups.The average age of the population was 69 years old, wherein 39.4% represented men, 39.8% represented non-whites, 64.9% had low education and 18.2% were smokers.There are also other characteristics shown in Table 1.In this sample, 8.4% of individuals could not perform at least one BADL-related task on their own.This rate increased to 13.7% for IADL-related tasks and 25.4% for mobility-related tasks.The ε4-carrier group displayed higher LDL-cholesterol levels, lower median CRP and a lower occurrence of elderly who were unable to walk without assistance.
The association between apoE genotypes, as well as the presence of ε4 allele and functional impairment in BADL, IADL and mobility-related tasks are shown in Tables 2 and 3. Association between ε4 allele presence and a better performance in mobility-related tasks could be detected when compared to reference genotype ε3/ε3  (OR = 0.66; 95%CI: 0.47-0.93)(Table 2) or when compared to non-ε4 carriers (OR = 0.65; 95%CI: 0.47-0.92)(Table 3), even after a complete adjustment for all confounding factors.The analysis considering the number of ε4 alleles showed similar results to those presented in Table 3, but the association between the presence of two alleles (ε4ε4) and disability in the mobility-related tasks was not statistically significant (OR = 0.61; 95%CI: 0.21-1.76),possibly due to the reduced number of homozygotes in this population (1.8%) (data not shown in the table ).

Discussion
This research showed a significant association between the presence of ε4 allele and lower odds of disability in the mobility-related tasks assessed, even after adjustment for confounding factors, in a population of the Brazilian elderly with low-education levels.Given the importance of functional status in healthy aging and the lack of studies addressing this kind of association, the results of the present study provide important information about this issue in an elderly population in Latin America.
The literature has shown inconsistent results about the influence of the apoE genotype in elderly functional performance.A case-control study conducted in a sample of Italian octogenarians and nonagenarians failed to detect an association between the apoE genotype and severe BADL disability, although the frequency of ε4-carriers was higher among those elderly that were considered "healthy".However, no confounding factor was considered in the analysis of the data 16 .A Taiwanese nested case-control study addressing the ε2 and ε4 allele presence influence and functional decline in BADL, IADL and mobility among elderly reported similar results.A genotype-dependent variation could not be found for this population when compared with individuals who presented functional decline and age and gender-matched controls.The analysis for this study was adjusted considering confounding factors 15 .Some North-American studies have reported a positive association between the presence of ε4 allele and disability.The first study to test this association was conducted in a sample of non-demented community-dwelling elderly who were free of stroke, Parkinson disease and depression 20 .It reported the presence of ε4 allele associated to greater odds of self-reported functional limitation in BADL, even after adjustment for confounding factors (age, gender, schooling and scores in neuropsychological tests).However another study found no significant association between the presence of ε4 allele and functional decline in BADL, IADL and physical health after four years of follow-up, although the authors reported interaction of the ε4 allele and female gender 18 .An interaction with gender among an elderly population of 627 living in a community was reported over a median follow-up of three years, and male elders carrying ε4 allele showed a greater risk of disability in BADL and a more rapid gait speed decline than male noncarriers 17 .In the Bambuí study, a gender interaction with the apoE genotype and functional performance was not found (data not shown).
Another study, conducted in a group of elderly people from the Netherlands, made use of performance tests and self-reported mobility disability for assessing functional status.For this Dutch research, the presence of ε4 allele was associated with poor performance in the five chair stand test but not in self-reports 19 .
A sub-sample of 1,805 elderly American participants who took part in the National Long-Term Care Survey (NLTCS) in 1999, showed that disability status is sensitive to apoE polymorphism, however, contrary to previous reports, this study reports that female ε4ε4 carriers have diminished chances of developing BADL disabilities (OR = 0.19; 95%CI: 0.40-0.99).On the other hand, the ε2ε3 genotype increased the chances of IADL impairments in males (OR = 2.33; 95%CI: 1.28-4.25) 14, and, heretofore, that was the first study to show a positive effect of ε4 allele in elderly functional performance.In the Bambuí study, although no association between the apoE genotype and BADL or IADL was detected, ε4carriers showed better completion in mobilityrelated tasks.
Many researchers have considered the ε4 allele to be the ancestral allele, which has been replaced by the ε3 allele along human evolution.In order to explain this hypothesis, it was postulated that the adverse effects of ε4 may have been balanced in an earlier time of higher prevalence of infectious diseases by increased host resistance, yet the adverse effects have been amplified because of the increase in longevity 32 .There is, indeed, a greater ε4 allele frequency in populations where the prevalence of infection and nutritional diseases is still high 33,34,35 .Studies conducted in a Brazilian shantytown located in Fortaleza, the capital city of the state of Ceará (in the Northeast of Brazil), found that early childhood diarrhea and enteric parasitic infections were less frequent in ε4-carriers, suggesting a protecting effect against parasitic infections 36,37 .The ε4 allele is considered a "thrifty" genotype, providing advantages in pathogen resistance, particularly in situations of malnourishment 38 .Considering that, in the past, living conditions in Bambuí were very close to living conditions of pre-industrialized populations, ε4-carriers may have taken advantage of this condition.
Another potential explanation for the results found in the present study is the fact that the presence of the ε4 allele leads to greater intestinal absorption and renal reuptake of vitamin D 39 .It has been suggested that higher concentrations of this vitamin may promote physical performance 40,41 , decreasing the risk of mobility limitation 42 .However, vitamin D concentration was not performed in the present study, making it difficult to evaluate this hypothesis.
It is also important to point out that in this same population, the presence of ε4 allele is associated with lower cognitive performance 43 , but we considered cognitive functioning to be a confounding factor.In addition, it has been shown that the cognitive deficit is more strongly associated with the decline of IADL (which was not associated with apoE genotypes in this study) compared with BADL and mobility 18 , showing that other factors may modulate this association, besides cognitive decline 44 .
The current study has its limitations, due to its cross-sectional design and the possibility of survivorship bias that would decrease the frequency of ε4-carriers in the sample, since this allele has been associated to mortality in some populations.In Bambuí, however, the apoE genotype did not predict mortality in the first nine years of follow up 11 , a fact that minimizes the in-fluence of survivorship bias in the data presented.Another limitation concerns the use of selfreports to assess functional performance, given that a previous study detected the impact of apoE polymorphism in tested mobility performance but not in self-reported mobility limitations 19 .Another point to be considered is the possibility of residual confounding, considering the wide range of mobility determinants (cognitive, psychosocial, physical, environmental, financial and personal life history) 44 , but it was not the aim of this study to explore these determinants.On the other hand, the main advantage of this study is that it concerns a population-based study conducted by a well-trained team that carefully collected all data using standardized protocols and followed strict quality control.It is also the first study to test an association between the apoE genotype and disability in a Latin American elderly population.It is therefore expected that this research will contribute to a better understanding in this field.
Given the above, it can be said, in short, that this study has shown the protective effect against mobility-related disabilities of the ε4 allele in a population living in a community, even when taking into consideration potential confounding factors.The reasons are not fully understood but may relate to its role as a thrifty gene in a sample exposed to high risk of infectious and nutritional diseases.It is also important to consider genegene and/or gene-environment interactions dealing with apoE and functional performance in the elderly.
ContributorsR.Z. Megale contributed to the study conception and design, analysis of data, and drafting of manuscript.A. I. Loyola Filho and M. F. Lima-Costa contributed to the study design and critical revision.J. O. A. Firmo contributed to the study design, acquisition of data and critical revision.S. V. Peixoto contributed to the study conception and design, analysis of data, drafting of manuscript and critical revision.

Table 1
Characteristics of the participants of the study, according to the apolipoprotein E genotype.Baseline of the Bambuí Health and Aging Study.

Table 2
Associations between apolipoprotein E genotypes and disabilities in mobility-related tasks, IADLs e BADLs.Baseline of Bambuí Health and Aging Study.

Table 3
Association between ε4 allele and disabilities in mobility-related tasks, IADLs e BADLs.Baseline of the Bambuí Health and Aging Study.