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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 61:918-923 (2006)
© 2006 The Gerontological Society of America

Sex Differences in the Association of Apolipoprotein E and Angiotensin-Converting Enzyme Gene Polymorphisms With Healthy Aging and Longevity: A Population-Based Study From Southern Italy

Davide Seripa, Marilisa Franceschi, Maria G. Matera, Francesco Panza, Patrick G. Kehoe, Carolina Gravina, Giuseppe Orsitto, Vincenzo Solfrizzi, Giovanni Di Minno, Bruno Dallapiccola and Alberto Pilotto

1 Laboratory of Geriatrics and Gerontology, Research Department, and 2 Geriatric Unit, Medical Sciences Department, IRCCS "Casa Sollievo della Sofferenza," San Giovanni Rotondo (FG), Italy.
3 Department of Geriatrics, Center for Aging Brain, Memory Unit, University of Bari, Italy.
4 Care of the Elderly, Department of Clinical Science at North Bristol, University of Bristol, United Kingdom.
5 Laboratory of Hemostasis and Thrombosis, Research Department, IRCCS "Casa Sollievo della Sofferenza," San Giovanni Rotondo (FG), Italy.
6 Department of Clinical and Experimental Medicine, "Federico II" University, Naples, Italy.
7 Research Department, IRCCS "Casa Sollievo della Sofferenza," San Giovanni Rotondo (FG), Italy.
8 CSS-Mendel Institute, Rome, Italy.

Address correspondence to Davide Seripa, PhD, Laboratory of Geriatrics and Gerontology, Research Department c/o Poliambulatorio "Giovanni Paolo II," IRCCS "Casa Sollievo della Sofferenza," Viale Cappuccini, San Giovanni Rotondo (FG), I-71013. E-mail: dseripa{at}operapadrepio.it


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
We investigated the association of sex and age with the occurrence of apolipoprotein E (apoE) and angiotensin-converting enzyme (ACE) genotypes in healthy aging and longevity in 1344 healthy individuals and 64 centenarians. As compared to participants younger than 60 years, a significant higher frequency of the apoE/{varepsilon}2 was observed in men aged 60–90 years (p <.001) and in centenarians (p <.001). Logistic regression analysis confirmed this outcome in both participants aged 60–90 years (odds ratio [OR] = 1.897; 95% confidence interval [CI], 1.227–2.931) and centenarians (OR = 3.263; 95% CI, 1.860–5.722). A further significant association of ACE/D allele and age was observed in centenarians (OR = 2.135; 95% CI, 1.253–3.636). Heterosis was also observed at the ACE locus. No relationship between apoE and ACE polymorphism was found. These findings suggest a role of sex in the association of apoE and ACE gene polymorphisms with healthy aging and longevity.

RECENT studies have indicated that longevity and the susceptibility to age-related diseases may be associated with different relative frequencies of common gene polymorphisms (1,2), such as apolipoprotein E (apoE) and angiotensin-converting enzyme (ACE) (3–7).

ApoE polymorphism is the result of a combination of two single nucleotide polymorphisms in exon 4 of the apoE gene forming three common alleles: {varepsilon}2, {varepsilon}3, and {varepsilon}4, which encode for the three protein isoforms, E2, E3, and E4, and six common genotypes (8). These apoE polymorphisms are associated with several age-related diseases such as Alzheimer's disease and vascular diseases (9), and seem to influence life expectancy (10). Thus, apoE polymorphism could be a marker for longevity and healthy aging (3,5).

ACE polymorphism involves the insertion of a 287-base-pair Alu element in intron 16 of the gene. This insertion generates allele I (Alu+), compared with the normal sequence defined as allele D (Alu–) (11). Although less investigated than apoE, ACE polymorphism has also been associated with several age-related diseases (12), as well as longevity (3,4,6). While sex-specific differences between both of these polymorphisms and age-related diseases have been reported (13,14), their role in healthy aging and longevity in Caucasians is not well defined. The aim of this study was to evaluate the association of sex and age with the frequency of apoE and ACE polymorphisms, and in particular the frequency of apoE/{varepsilon}2 and ACE/D alleles in a population-based sample of healthy persons and in a group of centenarians living in southern Italy.


   MATERIALS AND METHODS
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Study Samples
A total of 1710 individuals (757 men and 953 women, mean age 41.31 ± 13.63 years, range 18–90 years) were consecutively recruited among the personnel and relatives of the inpatients of the Istituto di Ricovero e Cura a Carattere Scientifico "Casa Sollievo della Sofferenza," San Giovanni Rotondo, Italy, as part of a research program investigating population genetics in Southern Italy. Only persons without a clinical history or evidence of neoplasms, infectious or inflammatory diseases, and/or acute disorders were initially considered for inclusion. After written informed consent was obtained, participants received a structured interview to evaluate the presence of age-related risk factors for degenerative diseases. Two hundred fifty-two individuals who had clinical evidence of vascular diseases (n = 215, hypertension, ischemic cardiac, and cerebrovascular diseases, peripheral vascular diseases, diabetes) or cognitive impairment (n = 37, Mini-Mental State Examination score lower than 26/30 points; 15) were excluded from the study. Moreover, 114 persons with serum levels of total cholesterol >220 mg/dL and/or low-density lipoprotein cholesterol >130 mg/dL and/or triglycerides >200 mg/dL (indicative of altered lipoprotein metabolism and increased risk of vascular diseases) were also excluded. Thus, a total of 1344 unrelated healthy Caucasians (79% of the overall population, 592 men and 752 women, mean age 41.31 ± 13.63 years, range 22–90 years) were included in the study. All participants were Caucasians originating from Apulia, southern Italy.

Additionally, a sample of 64 centenarians (18 men, 46 women; mean age = 100.28 ± 1.86, range 98–108 years) was recruited through the Central Population Register of Apulia and included in the study. They were all Caucasians residing in southern Italy. The inclusion criteria were: (a) an age of at least 98 years on the day of blood collection, (b) no known diagnosis of dementia or other chronic neurological diseases or psychiatric syndromes with cognitive impairment (16), and (c) no cerebrovascular disease, nephropathy, or end-stage renal disease, and no severe functional limitations.

ApoE and ACE Genotyping
Genomic DNA was manually purified from 4 mL of frozen blood samples by organic protein extraction and ethanol precipitation according to standard methods (17). The apoE and ACE genotypes were determined by polymerase chain reaction (PCR) and agarose gel electrophoresis as follows.

Briefly, the two transition C3937->T (Arg112->Cys) and C4075->T (Arg158->Cys) resulting in the apoE polymorphism, that is, the two single nucleotide polymorphisms rs429358 and rs7412, abolished two HhaI restriction sites (GCGC->GTGC), corresponding to codons 112 and 158 of the protein. We used the forward primer described by Houlston and colleagues (18) (TCC AAG GAG CTG CAG GCG GCG CA) and a reverse primer modified from Emi and colleagues (19) (CCC TCG CGG GCC CCG GCC TGG TAC AC). Primers were synthesized by Invitrogen (Invitrogen Corporation, Carlsbad, CA). A total of 100 ng of genomic DNA was amplified on an Applied Biosystems GeneAmp PCR System 9700 (Applied Biosystems Inc., Foster City, CA). Amplification conditions were 94°C for 2 minutes, followed by 35 cycles at 96°C for 30 seconds, 65°C for 45 seconds, and 72°C for 45 seconds. Reaction buffer included 1.5 U of Taq DNA polymerase (Platinum Taq; Invitrogen Corporation), 10 pmol of each primer, 100 µM of each deoxynucleotide triphosphate, and 1 mM MgCl2. Digestion of PCR product with 4 U of HhaI (New England Biolabs, Inc., Beverly, MA) at 37°C overnight produced a typical fragment pattern on a 4% agarose gel (20), one for each of the six apoE genotypes.

The ACE genotypes were identified as previously described (21). Briefly, the forward CTG GAG ACC ACT CCC ATC CTT TCT and the reverse GAT GTG GCC ATC ACA TTC GTC AGA T primers were used for PCR in a total of 30 cycles at 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 45 seconds. Primers were synthesized by Invitrogen (Invitrogen Corporation). Reaction buffer included 1 U of Taq DNA polymerase (Platinum Taq, Invitrogen Corporation), 10 pmol of each primer, 100 µM each deoxynucleotide triphosphate, and 1.5 mM MgCl2. Given the 287 bp size difference, a direct analysis of PCR products on a 1.8%–2.0% agarose gel identified the three ACE genotypes.

Statistical Analysis
Agreement of the observed genotype frequencies with the expected Hardy–Weinberg frequencies was verified with Arlequin Software, version 2.000 (22,23). Relative allele frequencies were estimated by the gene counting method (24), and the 95% confidence intervals (CIs) expressing variances of the estimated allele frequencies were also calculated (23).

We considered genotypes according to the presence of at least one allele. For the apoE polymorphism, we reported {varepsilon}2 ({varepsilon}2/) and {varepsilon}4 ({varepsilon}4/) carriers. {varepsilon}2 carriers were defined as those participants showing at least one {varepsilon}2 allele, that is, having a {varepsilon}2/{varepsilon}2, a {varepsilon}2/{varepsilon}3, or a {varepsilon}2/{varepsilon}4 genotype. Similarly, {varepsilon}4 carriers were defined as those participants showing at least one {varepsilon}4 allele, that is, having a {varepsilon}4/{varepsilon}2, a {varepsilon}4/{varepsilon}3, or a {varepsilon}4/{varepsilon}4 genotype. For ACE polymorphism we reported D (D/) and I (I/) carriers. D carriers were defined as those participants showing at least one D allele, that is, having a D/D or a D/I genotype. Similarly, I carriers were those participants showing at least one I allele, that is, having an I/I or an I/D genotype.

Pearson's chi-square test and Fisher's Exact test were used to compare genotype and estimated allele frequencies. Binary logistic regression analysis was used to verify the associations between apoE and ACE alleles with age and sex, as well as to verify interactions between apoE and ACE alleles. All statistical analyses were made with SPSS statistical software (version 10.1.3; SPSS Inc., Chicago, IL). A value of p <.05 was considered to be statistically significant.


   RESULTS
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
The overall study population of 1344 participants was divided into four groups according to age, as follows: Group 1 (n = 292, 21.73%), participants aged ≤ 30 years (range from 22 to 30 years of age, mean age 27.29 ± 2.10 years); Group 2 (n = 629, 46.80%), participants aged from 31 to 44 years (mean age 36.76 ± 3.94 years); Group 3 (n = 270, 20.09%), participants aged from 45 to 59 years (mean age 50.44 ± 4.17 years); and Group 4 (n = 153, 11.38%), participants aged from 60 to 90 years (mean age 70.71 ± 7.51 years). This distribution reflected that of the healthy population living in the Apulia region as reported by the ISTAT (Istituto Italiano di Statistica) Report 2002 (25).

ApoE genotype frequencies in the total sample of 1344 participants did not differ significantly from the expected Hardy–Weinberg frequencies (p =.364): The estimated allele frequencies were {varepsilon}2 = 0.060 (95% CI, 0.051–0.070), {varepsilon}3 = 0.836 (95% CI, 0.821–0.850), and {varepsilon}4 = 0.104 (95% CI, 0.093–0.116). Similarly, apoE genotype frequency in centenarians did not differ significantly from the expected Hardy–Weinberg frequencies (p =.422): The estimated allele frequencies were {varepsilon}2 = 0.148, {varepsilon}3 = 0.844, and {varepsilon}4 = 0.008.

When genotypes were compared among the different age groups (Table 1), a significantly higher {varepsilon}2 frequency was found in Group 4, that is, participants aged from 60 to 90 years, compared to participants younger than 60 years, that is, participants of Group 1 + Group 2 + Group 3 (19.61% vs 10.41%, p =.001). This finding was mainly due to a higher prevalence of {varepsilon}2 in Group 4 than in Group 3 (19.61% vs 9.26%, p =.002) and Group 2 (19.61% vs 9.38%, p <.001). No significant difference in {varepsilon}2 frequency was found between Group 4 and centenarians (19.61% vs 29.69%, p =.105). No participants showing genotypes {varepsilon}2/{varepsilon}2, {varepsilon}2/{varepsilon}4, or {varepsilon}4/{varepsilon}4 were observed in the centenarian group. In a linear model, no association was found between apoE genotypes and age.


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  		Table 1. Apolipoprotein E (ApoE) Genotypes in Relation to Sex and Age in 1344 Healthy Elderly Persons and 64 Centenarians.

 
In a sex x genotype analysis, no significant differences were found in the 1344 participants (p =.983) or in centenarians (p =.792). However, after dividing the participants according to sex, a significant difference in {varepsilon}2 frequency was found in men 60–90 years of age compared to men less than 60 years of age, that is, Group 4 versus Groups 1–3 (22.89% vs 9.63, p <.001), Group 4 versus Group 3 (22.89% vs 8.41%, p =.005), and Group 4 versus Group 2 (22.89% vs 7.72%, p <.001). Moreover, the {varepsilon}2 frequency found in men of Group 4 was quite similar to that found in centenarian males (22.89% vs 27.78%, p =.659). No differences were observed in {varepsilon}2 frequency in women (data not shown). No significant differences in {varepsilon}4 frequency were found across the age groups in the overall study sample or in separate groups of men and women (data not shown). Notably, only one participant (a woman) in the centenarian group had an {varepsilon}4 allele. Logistic regression analysis (Table 2) confirmed this outcome, showing a significant role of sex in the association of {varepsilon}2 and old age, that is, participants in Group 4 (crude odds ratio [OR] = 1.897; 95% CI, 1.227–2.931; sex-adjusted OR = 1.015; 95% CI, 0.530–1.944) and centenarians (crude OR = 3.263; 95% CI, 1.860–5.722; sex-adjusted OR = 4.096; 95% CI, 2.178–7.703).


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  		Table 2. Association of Apolipoprotein E (ApoE) Alleles With Age in 1344 Healthy Elderly Persons and 64 Centenarians.

 
The ACE genotype frequencies of the total 1344-participant group did not significantly differ from the expected Hardy–Weinberg frequencies (p =.299): Estimated allele frequencies were I = 0.349 (95% CI, 0.331–0.367) and D = 0.651 (95% CI, 0.633–0.669). Similarly, ACE genotype frequencies in centenarians did not differ significantly from the expected Hardy–Weinberg frequencies (p >.999): Expected allele frequencies were I = 0.313 and D = 0.688.

When genotypes were compared across age groups (Table 3), no significant differences were observed in the frequency of D participants in the oldest age group compared to participants less than 60 years of age, that is, Groups 1–3 (88.89% vs 86.99%, p =.507), Group 3 (88.89% vs 87.41%, p =.653), and Group 2 (88.89% vs 86.96%, p =.521). Furthermore, no difference was found in the frequency of D participants in the oldest age group compared to centenarians (88.89% vs 90.63%, p =.705). In a linear model, no association was found between ACE genotypes and age.


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  		Table 3. Angiotensin-Converting Enzyme (ACE) Genotypes in Relation to Sex and Age in 1344 Healthy Elderly Persons and 64 Centenarians.

 
In a sex x genotype analysis, no significant differences were found in the 1344 participants (p =.875) or in centenarians (p =.414). As expected, after dividing participants according to sex, no differences in the distribution of ACE allele among the age groups were found. Logistic regression analysis (Table 4) confirmed the lack of a significant association between the D allele and age. After adjustment for sex, however, a significant association between the D allele and age was observed in the oldest age group (crude OR = 1.177; 95% CI, 0.693–2.001; sex-adjusted OR = 0.453; 95% CI, 0.453–0.897) and centenarians (crude OR = 1.419; 95% CI, 0.603–3.338; sex-adjusted OR = 2.135; 95% CI, 2.135–3.636).


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  		Table 4. Association of Angiotensin-Converting Enzyme (ACE) Alleles With Age in 1344 Healthy Elderly Persons and 64 Centenarians.

 
Notably, heterosis was observed when ACE genotypes were classified according to sex. In fact, a significant association of both I and D alleles was shown in Group 1 (sex-adjusted OR values = 1.581; 95% CI, 1.211–2.064 and 1.357; 95% CI, 1.048–1.759, respectively, for I and D alleles), Group 2 (sex-adjusted OR values = 0.753; 95% CI, 0.600–0.945 and 0.775; 95% CI, 0.628–0.956, respectively, for I and D alleles), and Group 4 (sex-adjusted OR values = 0.669; 95% CI, 0.455–0.983 and 0.637; 95% CI, 0.453–0.897, respectively, for I and D alleles). No relationship was found between apoE and ACE alleles in the different age groups, even after adjustment for sex (Table 5).


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  		Table 5. Shared Frequencies of Apolipoprotein E (ApoE) and Angiotensin-Converting Enzyme (ACE) Alleles in a Cohort of 1344 Healthy Elderly Persons and 64 Centenarians.

 

   DISCUSSION
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
This study investigated the role of sex in apoE and ACE polymorphism and their association with healthy aging and longevity. We studied two genetically homogeneous groups of Caucasians participants who lived in southern Italy, that is, a cohort of healthy participants and a group of centenarians. In these populations, we found a significantly higher prevalence of the {varepsilon}2 allele in participants older than 60 years and in centenarians than in younger participants. These findings suggest a role of the {varepsilon}2 allele in healthy aging and longevity, and confirm previous studies reporting a significant increase in the {varepsilon}2 allele frequency with advancing age, including centenarians (26–28).

An association of the {varepsilon}2 allele with aging may be explained by two hypotheses. First, the progressively increased frequency of the {varepsilon}2 allele with aging may have been a consequence of the disappearance in our selected healthy participants of the {varepsilon}4 allele, which is reported to be linked to a higher risk of age-related diseases (9) and mortality (29). Indeed, {varepsilon}4 has been defined as a frailty allele (30). The second hypothesis suggests that {varepsilon}2 could be a longevity-promoting allele, functioning independently from other apoE alleles. This hypothesis is supported by our findings showing that the frequency of {varepsilon}3 did not change with advancing age, as would be expected if the {varepsilon}2 increase was due to {varepsilon}4 disappearance in the oldest age group. Indeed, an unchanged {varepsilon}3 allele frequency across age has been reported to be an important prerequisite in carrying out genetic association studies on aging (31).

Dividing participants according to sex, we observed that an increase of {varepsilon}2 occurred only in elderly and centenarian men and not in women. Given the observation that in Western countries women live longer than man do, a differential sex ratio among age groups changes the number of individuals investigated group-by-group, that is, with advancing age. Accordingly, we used sex as a covariate to correct this bias. Logistic regression analysis confirmed this sex-related increase of {varepsilon}2 with aging. To our knowledge, this is the first time that a sex-related interaction between apoE alleles and aging is reported in a healthy population and in centenarians. As this was a cross-sectional study, we cannot explain the biological role, if any, of the {varepsilon}2 allele in healthy aging men. Similarly, we cannot evaluate the impact of the described genetic associations and heterosis in the excluded persons. However, the increased {varepsilon}2 frequency observed in men can be, on the contrary, consistent with or explained by the association of {varepsilon}4 with the well-known higher vascular risk reported in men than in women (13,14). Nevertheless, the number of individuals included in this study, as well as their age distribution, reflected the healthy population of our regional area, thus warranting a lower type 1 error rate (false positive) and recruitment bias (28).

In this study, D allele frequencies were uniform in all age groups, including centenarians. This finding is in agreement with the majority of studies (28,32,33) that have tried unsuccessfully to replicate the previously reported association of increased ACE frequency with longevity by Schachter and colleagues (3). After adjustment for sex, however, a significant association between the D allele and centenarian age was found, thus suggesting a sex-dependent interaction of ACE alleles with longevity. It is clear that other factors influencing aging should be considered to be covariate in the logistic regression analysis. However, in considering a sample of healthy individuals, we greatly reduced the number of these variables.

Also heterosis (34), that is, the association of both I and D alleles with age, was observed in Groups 1, 2, and 4. Heterosis occurs when heterozygotes show either a greater or lesser association with a phenotypic trait than do homozygotes or the wild type. Until now, this genetic phenomenon has not been described in ACE polymorphism, and may prove to be a confounding factor in studies investigating the relationship between ACE polymorphism and age. Indeed, heterosis is common in humans, being demonstrated in up to 50% of all gene associations (35).

In disagreement with previous studies demonstrating a significant interaction between apoE and ACE alleles in patients with age-related diseases (36,37), no interaction between the two alleles was found in our population, indicating that these polymorphisms are probably independent factors in healthy aging and longevity. In view of these findings, the {varepsilon}2 and D alleles in healthy male and female elderly and/or centenarian persons most likely play independent, parallel roles in aging and human longevity. Further studies are needed to clarify their relationship with health and disease.


   Acknowledgments
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
This work was exclusively supported by grants from Ministero della Salute, Ricerca Corrente 2003-2005, Linea n. 1 "Malattie cardio e cerebrovascolari" and Ricerca Corrente 2006-2008, Linea n. 2 "Malattie di rilevanza sociale."


   Footnotes
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 Abstract
 Materials and Methods
 Results
 Discussion
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Decision Editor: James R. Smith, PhD

Received November 18, 2005

Accepted March 21, 2006


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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