The renin-angiotensin-aldosterone system (RAAS) also plays a significant role in the maintenance of cardiovascular homeostasis. The angiotensin-converting enzyme (ACE) is an important gene of the RAAS that has been evaluated in the pathogenesis of hypertension, coronary artery disease, heart failure, and recently of longevity. Furthermore, the nuclear factor kappa B (NFkB) gene is a regulator of the genes that encode cytokines and cell-adhesion molecules and induce the expression of growth factors and other factors involved in stress responses, cell proliferation, and cell cycle progression.10 Lee et al11 reported increased levels of NFkB in senescent cells in association with increased oxidative stress. Furthermore, Leychenko et al12 demonstrated that NFkB activation plays a role in mediating vascular endothelial growth factor and coor- dinates the hypertrophic response of cardiomyocytes during pressure overload-induced hypertrophy (hypertension and heart disease are influencing aging). Potentially, the NFkB gene should be involved in longevity as well, since it is an essential integrator of stress response and cell survival.13

Genotyping

Genotyping was performed specifically for research purposes. Extraction of genomic DNA was performed on leukocytes separated from whole blood using a standard method with Flex- iGene DNA kit (Qiagen, GmbH, Germany). The polymorphism -94ins/delATTG (rs28362491) within the NFKB1 gene was genotyped using polymerase chain reaction (PCR) and restric- tion fragment length polymorphism (RFLP) analysis, as described by Glas et al.15 The results of restriction analyses are 3 possible genotypes analyzed by electrophoresis on a 4% agar- ose gel stained with ethidium bromide. The ins/ins (ATTGA2/ ATTGA2) genotype is characterized by fragment sizes 93 base pairs (bp) and 23 bp, the del/del (ATTGA1/ATTGA1) geno- type by fragment size 112 bp, whereas the heterozygous ins/del has all the fragments.

The aim of the present study was to investigate any differ- ences in the frequency of common gene polymorphisms in non- agenarians, centenarians, and in middle-aged individuals (controls). Thus, we evaluated the single nucleotide poly- morphisms (SNPs) of CETP in chromosome 16 such as rs708272 (TaqB1) in position 56996288 (genotypes: B1B1, B1B2, B2B2) and rs5882 (I405 V) in position 57016092 (geno- types: II, IV, VV), ACE in chromosome 17 such as rs1799752 [Ins(I)/Del(D) polymorphism] in position 61565890 (geno- types: II, ID, DD), and NFkB in chromosome 4, such as rs28362491 [-94ins/delATTG (W/D)] in position 103422155 (genotypes: Ins/Ins, Ins/Del, Del/Del).

The ACE I/D polymorphism within the ACE gene was performed as described by Rigat et al.16 The PCR product is a fragment of 190 bp when the insertion is absent and 490 bp when the insertion is present; these were analyzed by electro- phoresis on a 4% agarose gel stained with ethidium bromide.

Statistical Analysis

Age was not normally distributed according to the Shapiro-Wilk test, and thus it is described as median and interquartile range (75th-25th percentile). All categorical variables are reported as absolute (n) and relative (%) frequencies. The associations between the studied variables were evaluated using chi-square test. All tests were 2-sided and the level of significance was set

Patients and Methods

The current study was designed and performed in agreement with the recommendations of the human genotype–phenotype

Table 2. CETP, ACE, and NFkB Genotype Frequencies of the Control and Nonagenarian Groups.

Table 1. Study Participants’ Characteristics Including CETP, ACE, and NFkB Genotype Frequencies.

n % Age (years) 18 to <80 105 34.2% (cid:2)90 to 98 190 61.9% (cid:2)99 to 111 12 3.9% Gender Men 122 39.0% Women 191 61.0% CETP TaqB1 B1B1 102 33.0% B1B2 147 47.6% B2B2 60 19.4% CETP I405V II 122 40.9% IV 144 48.3% VV 32 10.7% ACEI/D II 54 17.7% ID 134 43.9% DD 117 38.4% NFkB[-94ins/delATTG (W/D) ins/ins 106 47.7% ins/del 94 42.3% del/del 22 9.9%

% <80 (cid:2)90 Age (years) N % N % P CETP TaqB1 B1B1 38 36.2 63 32.1 .439 3.9% B1B2 45 42.9 99 50.5 B2B2 22 21.0 34 17.3 CETP I405V II 43 44.3 75 38.5 .584 IV 45 46.4 97 49.7 VV 9 9.3 23 11.8 ACE I/D II 18 18.0 35 17.7 .841 ID 46 46.0 85 42.9 DD 36 36.0 78 39.4 NFkB[-94ins/delATTG (W/D) Ins/Ins 48 49.5 55 46.6 .912 Ins/Del 40 41.2 51 43.2 Del/Del 9 9.3 12 10.2 Alleles CETP TaqB1 B1 121 57.6 225 57.4 .958 B2 89 42.4 167 42.6 CETP I405V 9.9% I 131 67.5 247 63.3 .318 geno- V 63 32.5 143 36.7 VV); ACE, ACE I/D II, ID, DD]); I 82 41.0 155 39.1 .662 Ins/Ins, D 118 59.0 241 60.9 NFkB[-94ins/delATTG (W/D) Ins 136 70.1 161 68.2 .674 Del 58 29.9 75 31.8

Abbreviations: CETP, cholesteryl ester transport protein (CETP TaqB1, geno- types: B1B1, B1B2, B2B2 and CETP I405 V, genotypes: II, IV, VV); ACE, angiotensin-converting enzyme (ACE I/D [Ins(I)/Del(D), genotypes: II, ID, DD]); NFkB, nuclear factor kappa B (NFkB[-94ins/delATTG (W/D), genotypes: Ins/Ins, Ins/Del, Del/Del]).

at .05. Data were analyzed using STATA statistical software (Version 9.0, Stata Corporation, College Station, Texas).

Abbreviations: CETP, cholesteryl ester transport protein (CETP TaqB1, geno- types: B1B1, B1B2, B2B2 and CETP I405 V, genotypes: II, IV, VV); ACE, angiotensin-converting enzyme (ACE I/D [Ins(I)/Del(D), genotypes: II, ID, DD]); NFkB, nuclear factor kappa B (NFkB[-94ins/delATTG (W/D), genotypes: Ins/Ins, Ins/Del, Del/Del]).

Results

There were no failures in sample collection, DNA acquisition, or genotyping procedures. Genotypic and allele frequencies did not differ by gender in any of the study groups (all P > .05). Table 1 shows the characteristics of the study participants including CETP, ACE, and NFkB genotype frequencies.

Limitations

No adjustment for inflation of type I error due to multiple comparisons was made as a result of their relatively low number.