Invest Clin 64(3): 281 - 295, 2023 https://doi.org/10.54817/IC.v64n3a2

Corresponding author: Carolina Pestana. Laboratorio de Genética Molecular Humana B, Universidad Simón Bolí-

var, Valle de Sartenejas, Miranda, Venezuela, Phone: +58 (212) 906 4221. E-mail: carolinapestana@usb.ve

Relationships between genetic vascular risk

polymorphism and aging. A case-control

study in Venezuela.

Carlos Álvarez

, Andrea Bullones

, María A Medina

, Anna Vargas

, Antonietta Porco

Juan C Méndez

and Carolina Pestana

Laboratorio de Genética Molecular Humana B, Universidad Simón Bolívar, Valle de

Sartenejas, Miranda, Venezuela.

Academia Latinoamericana Antienvejecimiento, Miranda, Venezuela.

Keywords: aging; cardiovascular homeostasis; lipid metabolism; polymorphism; blood

coagulation.

Abstract: Aging is an irreversible process that produces the progressive

decline of physiological functions favoring the development of cardiovascular

complications associated with genetic Risk Alleles (RA). A case-control study

using a sample of 90 Venezuelan individuals was performed to determine the

correlation between the incidence of accelerated aging for 14 polymorphisms in

genes associated with blood coagulation, lipid, and cardiovascular homeostasis.

Odds Ratio (OR) results showed a 41% increase in the risk of presenting accel-

erated aging in subjects with the rs1800790 RA in the FGB gene. The CC geno-

type for the rs1800775 in the CETP gene was associated with a 62%, and the

TT genotype for the rs1801133 in the MTHFR gene increased risk by two times.

However, none of these results were statistically significant. Only a significant

association was determined between the presence of the homozygous deletion

genotype for the rs4340 RA in the ACE gene with an increased risk up to ten

times (OR: 10.6; CI: 1.23 - 90.67; p<0.05). Multivariable analyses showed that

gender, obesity, hypercholesterolemia, hypertriglyceridemia, smoking, age,

body mass index, systolic hypertension, the rs662 RA in the APOB, rs693 RA in

the PON1 and rs1801133 RA in the MTHFR genes were the main environmental

and genetic factors associated with accelerated aging.

282 Álvarez et al.

Investigación Clínica 64(3): 2023

Relación entre polimorfismos de riesgo genético vascular

y el envejecimiento. Un estudio caso-control en Venezuela.

Invest Clin 2023; 64 (3): 281 – 295

Palabras clave: envejecimiento; homeóstasis cardiovascular; metabolismo lipídico;

polimorfismo; coagulación sanguínea.

Resumen: El envejecimiento es un proceso irreversible que produce el

declive progresivo de las funciones fisiológicas favoreciendo el desarrollo de

complicaciones cardiovasculares asociadas con alelos de riesgo (AR) genéticos.

Se realizó un estudio caso-control empleando una muestra de 90 individuos ve-

nezolanos para determinar la correlación entre la incidencia de envejecimiento

acelerado para 14 polimorfismos en genes asociados a coagulación sanguínea,

lípidos y homeóstasis cardiovascular. Resultados de razón de probabilidades

(RP) mostraron en un 41% de sujetos con el AR rs1800790 en el gen FGB un

incremento en el riesgo de presentar envejecimiento acelerado. El genotipo CC

para el rs1800775 en el gen CETP fue asociado con un incremento de riesgo de

62% y el genotipo TT para el rs1801133 en el gen MTHFR con un incremento

en el riesgo de 2 veces. Sin embargo, ninguno de estos resultados fue estadísti-

camente significativo. Sólo se determinó una relación estadísticamente signi-

ficativa entre la presencia del genotipo de deleción homocigota para el AR de

rs4340 en el gen ACE con un riesgo incrementado de hasta 10 veces (RP: 10,6;

CI: 1,23 – 90,67; p<0,05). Los análisis multivariable mostraron que el género,

obesidad, hipercolesterolemia, hipertrigliceridemia, hábito tabáquico, edad, ín-

dice de masa corporal, hipertesión sistólica, el AR rs662 en el gen APOB, el AR

rs693 en PON1 y el AR rs1801133 en el gen MTHFR eran los principales facto-

res ambientales y genéticos asociados a envejecimiento acelerado.

Received: 12-12-2022 Accepted: 01-04-2023

INTRODUCTION

Aging is a complex time-depending

process that causes the progressive decline

of the organism’s physiological function, af-

fecting the cells’ adaptability and their abil-

ity to maintain homeostasis and leading to

a general decline of all body systems. There-

fore, aging increases the susceptibility of the

organism to suffer various diseases, usually

related to cellular senescence

1,2

. Aging is a

multifactorial process in which the interac-

tion of several genetic and environmental

variables can determine the growing old rate

among different individuals or between or-

gans and tissues of one specific individual

Specific genotypes appear to be associated

with accelerating the depletion of the organ-

ism’s metabolism leading to the premature

presence of degenerative diseases and the

stimulation and acceleration of the natural

aging course

Alterations in the coding genes of pro-

teins associated with endothelial function,

blood coagulation, and lipid metabolism

have proven to be highly related to the ap-

pearance of cardiovascular diseases (CVDs),

known pathologies related to aging. More-

Relationships between genetic vascular risk polymorphism and aging in Venezuela 283

Vol. 64(3): 281 - 295, 2023

over, several alleles for some polymorphic

variants in different candidate genes related

to vascular risk have been postulated as ge-

netic markers of premature aging

5,6

, and

many studies have been dedicated to estab-

lishing genetic variants which might be as-

sociated with healthy aging and longevity

7-12

. As a result, multiple variants in genes

involved in different cellular processes and

metabolic pathways have been postulated

8,11

with special attention on genes related to

CVDs due to the close relationship between

these diseases and aging

. However, due to

the complexity of the aging process, there

are some inconsistencies in the relation-

ships proposed regarding the genetic factors

involved in aging

10,11,14

, probably because

the environmental factors also play an es-

sential role in aging progression as they can

modulate the influence of the genetic risk

factors

13,15

Here, we evaluated the relationship be-

tween accelerated aging and different geno-

types of 13 polymorphisms in the following

genes: Apolipoprotein B (APOB; rs693), Apo-

lipoprotein E (APOE; rs429358 and rs7412),

Cholesteryl Ester Transfer Protein (CETP;

rs1800775), Paraoxonase 1 (PON1; rs662),

Fibrinogen Beta Chain (FGB; rs1800790

and rs1800791), Coagulation Factor II (F2;

rs1799963), Coagulation Factor V (F5;

rs6025), Coagulation Factor VII (F7; rs6046),

Methylenetetrahydrofolate Reductase (MTH-

FR; rs1801133), Angiotensin Converting En-

zyme (ACE; rs4340) Angiotensinogen (AGT;

rs699), and Nitric Oxide Synthase 3 (NOS3;

rs1799983) in a selected sample of 90 sub-

jects from Caracas, Venezuela.

These gene variants have been correlat-

ed to diseases linked to aging, such as CVDs

5,6

, and some of these variants have also been

associated with longevity

2,4,6

. Knowledge of

the genetic factors that may influence the

susceptibility of an individual to develop

CVDs would help identify, prevent, or slow

down the disease. Additionally, early diagno-

sis of these diseases can be used to plan per-

sonalized treatments to avoid the progres-

sion of these and other diseases and favor

healthy aging.

MATERIALS AND METHODS

Subjects

The sample comprised 90 randomly se-

lected individuals unrelated to the “Centro

Médico Antienvejecimiento” (CMA, Caracas,

Venezuela), whose biological age was deter-

mined. This sample was classified into two

groups: i) 30 control individuals, in which

the biological age was equal or under the

chronological age, and ii) 60 patients, which

were subdivided into 30 patients with ag-

ing grade 1 (G1), in which the biological

age was between 1 and 14 years over their

chronological age, and 30 patients with ag-

ing grade 2 (G2), in which the biological

age was between 15 and 28 years over their

chronological age.

Determination of the biological age

The biological age was estimated us-

ing various biological and anthropometrical

parameters such as body weight, body mass

index (BMI), body fat percentage, stimuli re-

sponse time, accommodation reflex, static

balance, skin elasticity, and blood pressure.

By comparing the estimated biological age

with the chronological age, which corre-

sponds to the time that has passed since the

individual´s birth, the subjects were classi-

fied into different aging grade groups

Blood Sampling

Peripheral blood was collected from all

subjects after obtaining their signed con-

sent. A standard proforma was filled up with

their personal information, having particu-

lar emphasis on age, gender, smoking habit

(current smokers or non-smokers), pres-

ence of hypertension (defined as a systolic

blood pressure of at least 140 mm Hg and/

or diastolic blood pressure of at least 90 mm

Hg), diabetes mellitus (defined by a blood

glucose level of at least 6.93 mmol/L) and

obesity (BMI over 30).

284 Álvarez et al.

Investigación Clínica 64(3): 2023

Genes and Polymorphisms Studied

We evaluated the relationship between

accelerated aging and different genotypes

of 13 polymorphisms in the following genes:

Apolipoprotein B (APOB; rs693), Apolipo-

protein E (APOE; rs429358 and rs7412),

Cholesteryl Ester Transfer Protein (CETP;

rs1800775), Paraoxonase 1 (PON1; rs662),

Fibrinogen Beta Chain (FGB; rs1800790

and rs1800791), Coagulation Factor II

(F2; rs1799963), Coagulation Factor V

(F5; rs6025), Coagulation Factor VII (F7;

rs6046), Methylenetetrahydrofolate Re-

ductase (MTHFR; rs1801133), Angiotensin

Converting Enzyme (ACE; rs4340), Angio-

tensinogen (AGT; rs699), and Nitric Oxide

Synthase 3 (NOS3; rs1799983).

Genotyping

Genomic DNA was extracted from total

peripheral blood as described by Bowen y

Keeney

. Details regarding identifying the

polymorphisms for every specific gene are

presented in Table 1.

Thirty cycles were performed follow-

ing a denaturation step at 94°C for 5 min.

Each cycle consisted of incubations at 94°C

for 1 minute, annealing temperature for 1

minute, and 72°C for 1 minute. A final ex-

tension step was carried out at 72°C for 10

min. PCR products were analyzed by elec-

trophoresis on a 2.5% agarose gel contain-

ing SYBR Safe. Gel images were document-

ed by using a digital camera equipped with

ultraviolet filters.

The enzymatic digestions were carried

out overnight at 37°C, and the digested sam-

ples were separated using an 8% polyacryl-

amide gel and visualized by silver staining

Statistical Analysis

Values of continuous variables were ex-

pressed as means ± standard deviations (SD).

The allelic frequency and the frequency of

heterozygous and homozygous carriers of the

studied polymorphisms were calculated in ev-

ery subject group (control, G1, and G2). The

number of cases and control subjects with a

specific genotype was used to determine the

risk, estimated as the Odds Ratio (OR) us-

ing the software PAST version 2.17c (2013)

in both a recessive and dominant model. The

OR represents the probability that the pres-

ence of accelerated aging occurs or not when

we compared the patients with the control

individuals, and it is defined as the ratio of

occurrence of accelerated aging between the

two groups

. Multivariable logistic curve re-

gression analyses were used to monitor the

risk of developing vascular disease as a result

of accelerated aging under various condi-

tions: genotype, age, gender, obesity, weight,

body fat percentage, BMI, smoking, presence

of hypertension, hypertriglyceridemia, hyper-

cholesterolemia, and diabetes mellitus. The

regression coefficients that were obtained

represented the probability of suffering the

disease because of the presence of the risk al-

lele of the polymorphisms and the other vari-

ables studied. Statistical significance was set

up at a p ≤ 0.05.

RESULTS

General characteristics

The general and biological character-

istics of the subjects conforming to the ag-

ing patient subgroups and control group are

shown in Table 2. The patient subgroup 2

(G2) was mainly composed of young individ-

uals, considering that the average chrono-

logical age (37.33 ± 11.8) was smaller than

the other groups. The G2 group contained a

higher percentage of individuals with smok-

ing habits, diabetes, and obesity, as well

as high blood pressure values. However, a

higher percentage of individuals with hyper-

cholesterolemia was observed in the control

group (Control).

Genotyping

Except for the polymorphism in the

APOB gene, all alleles and genotypes in the

control group were within the Hardy-Wein-

berg equilibrium (data not shown).

Relationships between genetic vascular risk polymorphism and aging in Venezuela 285

Vol. 64(3): 281 - 295, 2023

Table 1

Detection techniques employed to determine genotype in the different polymorphism studied.

Gene Polymorphism Variant

Type

Detection

Technique

Primers Sequence Ta Possible

Alleles

Reference

APOB rs693 SNV PCR-RFLP 3´-GATGAAACCAATGACAAAATCC-5´ 58°C G/A 19

3´-AACAGTGAACCCTTGCTCTACC-5´

APOE rs429358 SNV PCR-RFLP 3´-AGACGCGGGCACGGCTGTCCAAGGA-5´ 62 °C T/C 19

3´-CCCRCG CGGGCCCCGGCCTGGTACAC-5´

APOE rs7412 SNV PCR-RFLP 3´-AGACGCGGGCACGGCTGTCCAAGGA-5´ 62°C C/T 19

3´-CCCRCGCGGGCCCCGGCCTGGTACAC-5´

CETP rs1800775 SNV PCR-RFLP 3´- AGAATTGAAATGCCACAGACATTCC-5´ 57°C T/C 20

3´-CCTTGATATGCATAAAATAACTCTGG-5´

PON1 rs662 SNV PCR-RFLP 3´- TTGAATGATATTGTTGCTGTGGGACCTGAG-5´ 65°C T/A/

C/G

3´-CGACCACGCTAAACCCAAATACATCTCCCAGAA-5´

FGB rs1800790 SNV PCR-RFLP 3´- GGTCTTTCTGATGTGTATT-5´ 55°C G/A 20

3´-CTATTATTCTTTCTTGGTCTA-5´

FGB rs1800791 SNV PCR-RFLP 3´-GTGTTCCTATTGATTCTTCTTGTAGG-5´ 55°C G/A 20

3´-AATGAGGCCCATTTTCCTTGAAATT-5´

AGT rs699 SNV PCR-RFLP 3´-GATGCGCACAAGGTCCTCTG-5´ 61°C T/C 19

3´-CAGGGTGCTGTCCACACTGGCTCGC-5´

F7 rs6046 SNV PCR-RFLP 3´-CAGTCACGGMAGGTGGGAGAC-5´ 56°C G/A/

C/T

3´-GGGGTAATTGACGTCTTCTT-5´

MTHFR rs1801133 SNV PCR-RFLP 3´-GCCTCTCCTGACTGTCATCC-5´ 61°C C/T 21

3´-CCCTTTTGGTGATGCTTGTT-5´

NOS3 rs1799983 SNV PCR-RFLP 3´-CATGAGGCTCAGCCCCAGAAC-5´ 59°C G/T 22

3´-AGTCAATCCCTT TGGTGCTCAC-5´

ACE rs4340 Indel PCR 3´-CTGGAGAGCCACTCCCATCCTTTCT-5´ 58°C Ins/ Del 19

3´-GACGTGGCCATCACATTCGTCAGAT-5´

3´-TGGGACCACAGCGCCCGCCACTAC-5´ * 67°C

3´-TCGCCAGCCCTCCCATGCCCATAA-5´ *

F2 rs1799963 SNV ASPCR 3´-CACTGGGAGCATTGAGGCGC-5´ 59°C G/A 19

3´-ATGAATAGCAATGGGAGCATTGAGGATT-5´

3´-ATGTGTTCCGCCTGAAGAAGTGGA-5´

3´-CCCACCTTCCCCTCTCTCCAGGCAAATGGG-5´ **

3´-GGGCCTCAGTCCCAACATGGCTAAGAGGTG-5´ **

F5 rs6025 SNV ASPCR 3´-CAAGGACAAAATACCTGTATTCAT-5´ 58°C C/A/T 19

3´-CAAGGACAAAATACCTGTATTCTTT-5´

3´-GGCAGGAACAACACCATGAT-5´

3´-CCCACCTTCCCCTCTCTCCAGGCAAATGGG-5´ **

3´-GGGCCTCAGTCCCAACATGGCTAAGAGGTG-5´ **

Abbreviations: PCR-RFLP: Polymerase Chain Reaction-Restriction Fragment Length Polymorphism; PCR: Polymera-

se Chain Reaction; SNV: Single Nucleotide Variant; In/Del: Insertion-Deletion; ASPCR: Allele Specific Polymerase

Chain Reaction; Ta: Annealing Temperature. *Used for genotype verification; **Used as internal control.

286 Álvarez et al.

Investigación Clínica 64(3): 2023

The frequency of the risk allele of the

rs1800790 polymorphism (f: 0.15; CI: 0.13

– 0.17) in the FGB gene, rs1799963 poly-

morphism (f: 0.02; CI: 0.03 – 0.01) in the

F2 gene, rs6025 polymorphism (f: 0.03; CI:

0.05 – 0.02) in the F5 gene, rs1801133 poly-

morphism (f: 0.30; CI: 0.27 – 0.33) in the

MTHFR gene, and rs4340 polymorphism (f:

0.45; CI: 0.42 – 0.48) in ACE gene was more

significant in the G2 group than in the con-

trol group. The frequency of the risk allele

of the rs1799983 polymorphism (f: 0.30; CI:

0.245 – 0.354) in the NOS3 gene and rs662

polymorphism (f: 0.37; 0.34 - 0.40) in the

PON1 gene was higher in the G1 group than

in the control group. Finally, the risk allele

frequency of the rs429358 and rs7412 poly-

morphisms in the APOE gene was greater in

Table 2

General and biological characteristics of the population.

Variables Control (n=30) G1 (n=30) G2 (n=30)

Chronological age (X ± SD) 55.07 ± 8.08 50.43 ± 8.52 37.33± 11.8

Differential age (Biological age-

Chronological age) (X ± SD)

6.58 ± 4.89 6.87 ± 3.94 21± 10.8

Mode (years) 59 48 27

Female (%) 83.3 90 70

Presence of smoking habits (%) 30 33.3 43.3

Presence of diabetes (%) 0 0 6.7

Presence of obesity (%) 0 26.7 36.7

Presence of hypercholesterolemia (%)

43.3 40 23.3

Presence of hypertriglyceridemia (%)

16.7 26.7 23.3

Body weight (Kg) 58.95 ± 8.25

(n=29)

69.18 ± 13.62

(n=30)

74.27 ± 18.81

(n=30)

Body fat percentage (%) 28.84 ± 13.28

(n=29)

37.93 ± 9.99

(n=29)

36.27 ±11.67

(n=30)

Body Mass Index (kg/m

) 22.91 ± 2.98

(n=29)

26.34 ± 4.04

(n=27)

27.21 ± 5.42

(n=29)

Stimuli response time* (cm) 17.93·20.14·22.54

(n=28)

15.40·19.43·17.80

(n=30)

15.48·16.24·19.21

(n=29)

Accommodation reflex** (cm) 20.76 ± 8.15

(n=29)

21.07 ± 10.26

(n=29)

17.48 ± 4.65

(n=29)

Static balance *** (s) 7.86 · 5.90 · 7.14

(n=29)

4.45 · 4.45 · 6.10

(n=29)

6.07 · 4.03 · 5.93

(n=29)

Skin elasticity (s) 29.02 ± 41.65

(n=29)

12.28 ± 23.32

(n=30)

6.24 ± 12.37

(n=30)

Blood pressure (mmHg) Systolic/

Diastolic

124.86/79.21

(n=29)

127.21/77.86

(n=29/28)

132.93/81.50

(n=30)

Values came from answering “yes” in the questionnaire regarding the presence of hypercholesterolemia and hyper-

triglyceridemia. n<30 indicates that results couldn’t be obtained for all individuals for a specific variable while all the

others consider the 30 individuals’ population. *Values resulted from three measurements of the response speed of

the upper extremities due to visual stimuli. **Values represent the distance between the eye and the text when the

text can still be focus correctly. ***Time in which the patient started to oscillate or swing with the eyes close.

Relationships between genetic vascular risk polymorphism and aging in Venezuela 287

Vol. 64(3): 281 - 295, 2023

the control group (f: 0.21; CI: 0.16 - 0.25)

than in the other groups.

Figura 1 shows schematically the OR

value calculated for each polymorphic vari-

ant in the G1 and G2 subgroups. The asso-

ciated risk calculated using the OR for the

presence of the risk allele of the rs1800790

polymorphism in the FGB gene showed a

41% higher tendency to exhibit an acceler-

ated aging (OR: 1.41; CI: 0.44-4.45) (Fig.

1), but these results were not statistically

significant (p>0.05). Regarding the risk al-

lele of the rs1799963 polymorphism in the

F2 gene and of the rs6025 polymorphism in

the F5 gene, we were unable to determine

the OR value since the risk allele was only

present in the G2 group. However, these

two polymorphisms have been established

as independent factors for vascular risk.

Moreover, the CC genotype for the

rs1800775 polymorphism in the CETP gene

was associated with a 62% increased risk of

accelerated aging (OR: 1.62; CI: 0.40- 6.40;

p>0.05). The calculated OR value for the

rs662 polymorphism in the PON1 gene sug-

gested that the occurrence of the risk allele

is associated with a 15% increase in the risk

of having accelerated aging (OR: 1.15; CI:

0.4-3.26; p>0.05) (Fig. 1). Also, the pres-

ence of the risk allele of the APOE gene

was not associated with an increased risk of

showing an accelerated aging (Fig. 1). All

these results were not statistically signifi-

cant (p>0.05).

Regarding the presence of the TT geno-

type for the rs1801133 polymorphism in the

MTHFR gene, it was observed an increase

by two times in the risk of exhibiting ac-

celerated aging (OR: 2.07; CI: 0.18-24.15;

p>0.05), however, this result was not statis-

tically significant.

Similarly, the presence of the Del al-

lele for the rs4340 polymorphism in the ACE

gene was also associated with a two-time in-

crease in the risk of exhibiting accelerated

aging (OR: 2.07; CI: 0.18-24.15; p>0.05),

showing no statistical significance. The risk

was increased up to ten times (OR: 10.6; CI:

1.23-90.67; p<0.05) by the presence of the

homozygote genotype for the risk allele dis-

playing statistical significance.

The presence of the CC genotype for

the rs699 polymorphism in the AGT gene

was associated with a two time-increase in

the risk of exhibiting accelerated aging (OR:

2.13; CI: 0.62-7.39; p>0.05) being this re-

sult not statistically significant.

Lastly, the presence of the T allele for

the rs1799983 polymorphism in the NOS3

gene was associated with a 96% increase in

Fig. 1. OR values calculated comparing the control group with the G1 (Left) and G2 (Right) groups; with

every value it shows the error bars which represent the confidence interval in each case.

288 Álvarez et al.

Investigación Clínica 64(3): 2023

the risk of exhibiting accelerated aging (OR:

2.13; CI: 0.62-7.39; p>0.05) being this re-

sult not statistically significant.

A multivariable statistical analysis was

carried out to determine the association

between the development of accelerated

aging and each one of the genetic and envi-

ronmental variables that define the sample.

Table 3 shows the variables that were taken

into consideration for the analysis. In the

G1 subgroup, the environmental and genet-

ic factors that proved to be associated with

the development of an accelerated aging

process were obesity, hypercholesterolemia

and hypertriglyceridemia, age, BMI, APOE

rs429358 and rs7412 polymorphisms, CETP

rs1800775 polymorphism, FGB polymor-

phism rs1800790 and MTHFR polymorphism

rs1801133. In the G2 subgroup, the environ-

mental and genetic factors associated with

accelerated aging were age, sex, body mass

index, hypertriglyceridemia, smoking, systol-

ic hypertension, F7 polymorphism rs6046,

and MTHFR polymorphism rs1801133.

DISCUSSION

Recent studies in Latin American popu-

lations have shown the existence of differen-

tial ancestral contribution patterns between

and within groups, which correlate with

the indigenous population density before

the conquest of America and with the cur-

rent demographic growth patterns in these

regions

. This agrees with genetic studies

carried out in Venezuela, based on the analy-

sis of blood group polymorphisms and DNA

polymorphisms, which have revealed that, as

in other Latin American countries, the con-

quest and colonization processes generated

very heterogeneous populations. In general,

the genetic component that prevails in these

studies is the Mediterranean European, fol-

lowed by the indigenous and, to a lesser ex-

tent, the African, and also with a marked

inter- and intra-regional difference

. Spe-

cifically, for the population of Caracas, in a

study carried out by Martínez et al.

, who

performed the analysis of five autosomal

markers found in the high socioeconomic

stratum, the European component (0.78)

was found in a higher proportion than Sub-

Saharan African, which was almost negligible

(0.06); while for the low socioeconomic lev-

el, the Sub-Saharan, European, and Amerin-

dian components were 0.21, 0.42 and 0.36,

respectively. Therefore, to conduct genetic

studies in a highly heterogeneous popula-

tion like ours, it is imperative to understand

the high degree of genetic variability of the

different ethnic groups that inhabit the ter-

ritory.

Attributable to the impact of the ge-

netic constitution of any individual in the

development of a particular phenotype, the

sum of the genetic alterations or risk alleles

of different genes can help us elucidate the

effect of these in the evolution of the disease

through molecular diagnosis. Likewise, the

molecular diagnosis of risk alleles associated

with vascular risk can help supplement the

results of biochemical and clinical analyses

and therefore provide an answer or an expla-

nation to a disease or any given family his-

tory.

With the multivariable analysis we con-

firmed and demonstrated that age is directly

related to accelerated aging, because as the

years pass by diminishes the organism capac-

ity to maintain homeostasis causing tissue

failure and malfunction of the regulation

systems, which in turn may produce an in-

crease in the susceptibility to suffer various

diseases. We also found that hypercholes-

terolemia and hypertriglyceridemia are risk

factors associated with the development of

accelerated aging, suggesting that the pres-

ence of high levels of cholesterol and triglyc-

erides promote the rapid deterioration of

the organism, mainly at the vascular level,

given that high values of cholesterol and tri-

glycerides are related to the formation of

atheroma and the unfolding of atherosclero-

sis

27,28

Other variables associated with an ac-

celerated process of aging were obesity and

Relationships between genetic vascular risk polymorphism and aging in Venezuela 289

Vol. 64(3): 281 - 295, 2023

Table 3

Association between the different genetic and environmental factors and the development

of vascular risk through an accelerated aging.

Variable Association with G1 (n=60) Association with G2 (n=60)

Sex Not Associated Associated*

Diabetes mellitus -- Not Associated

Obesity Associated* Not Associated

Hypercholesterolemia Associated** Not Associated

Hypertriglyceridemia Associated* Associated*

Smoking Not Associated Associated*

Age Associated** Associated***

Weight Associated** Not Associated

Body fat percentage Not Associated Not Associated

BMI Not Associated Associated***

Systolic hypertension Not Associated Associated***

Diastolic hypertension Not Associated Not Associated

APOB (rs693)

Allele 7545T

Associated* Not Associated

APOE (rs429358 y rs7412)

Allele 388C y 526C

Not Associated Not Associated

CETP (rs1800775)

Allele -656C

Not Associated Not Associated

PON1 (rs662)

Allele 575G

Associated* Not Associated

FGB (rs1800790)

Allele -455A

Not Associated Not Associated

FGB (rs1800791)

Allele -854ª

Not Associated Not Associated

F2 (rs1799963)

Allele 20210A

-- Not Associated

F5 (rs6025)

Allele 1691A

-- Not Associated

F7 (rs6046)

Allele 10976G

Not Associated Not Associated

MTHFR (rs1801133)

Allele 655T

Not Associated Associated*

ACE (rs4340)

Allele Del

Not Associated Not Associated

AGT (rs699)

Allele 803C

Not Associated Not Associated

NOS3 (rs1799983)

Allele 894T

Not Associated Not Associated

* p< 0,05; ** p< 0,01; *** p< 0,001; -- excluded from the model.

290 Álvarez et al.

Investigación Clínica 64(3): 2023

BMI, being BMI a vascular risk indicator that

is used universally to detect overweight and

obesity. Obesity promotes the appearance

of alterations in various mechanisms of hor-

monal regulation, being hyperinsulinemia

and leptinemia, some of the most common

hormonal alterations associated with obe-

sity

. Moreover, obesity accelerates the ag-

ing of adipose cells increasing the formation

of reactive oxygen species in fat cells, pro-

moting inflammatory processes and insulin

resistance. Aging and obesity not only favor

the deregulation of the metabolism but also

promote the development of hypertension,

dyslipidemia, and cardiovascular complica-

tions

29,30

Hypertension is known as a risk factor

for the development of cardiovascular dis-

ease. In the present study, our results sug-

gest that it is associated with accelerated

aging, meaning that the deterioration of

the vascular endothelium due to high blood

pressure constitutes a significant risk factor

for accelerating this process

Regarding the allelic variant 20210A

of the rs1799963 in the F2 gene, while an

association could not be established in this

investigation, this gene has been linked

with premature aging, being reported with

a decreased frequency in the middle to ad-

vanced-age individuals

. In turn, the allelic

variant 1601A of the rs6025 polymorphism

in the F5 gene has been associated as an

independent factor that indicates blood hy-

per-coagulation.

The presence of this risk allele causes

the production of a factor V protein that

cannot be degraded by the activated pro-

tein C (APC), and consequently, an increase

in the amount of factor V is obtained in the

blood

. It is worth noting that similarly to

rs1799963 in F2, the allelic frequency for

the 1601A variant in F5 has been reported

to decrease in individuals with advanced

age (centenary individuals)

. Regarding

the rs6046 polymorphism in the F7 gene,

the presence of the mutant allele 1172A is

beneficial for the prevention of cardiovas-

cular diseases or the formation of vascular

thrombus since this mutant allele produces

a protein that has a deficient interaction

with the tissue factor, causing a lower ini-

tiation of the secondary hemostasis

Concerning the genes involved in lipid

metabolism, it has been reported that the

presence of the allele E4 in the APOE gene

promotes the appearance of cardiovascular

diseases, which is why many studies address

the relationship between this gene and ag-

ing

7,8

. Although the OR values obtained here

do not suggest that this risk allele is associ-

ated with accelerated aging, the multivari-

able analysis showed that the allele E4 is one

of the risk factors that participate in the fast

deterioration of the organism. This allele

has been shown to decrease its frequency in

advanced age centenary groups; in turn, the

allele E2 which has been reported to have

a protective effect, has been reported to

increase in these groups, suggesting an as-

sociation with the aging process

. The dis-

crepancy might be because the multivariable

analysis considers the interaction of various

genetic variables in conjunction with envi-

ronmental variables related to aging.

The risk allele -656C for the rs1800775

polymorphism in the CETP gene was found to

be associated with accelerating aging. This

polymorphism is linked to the transcription

levels of the CETP gene, whose protein prod-

uct is involved in the transference of choles-

terol and other lipids from HDL to LDL and

VLDL

. Thus, the risk allele -656C, which is

the ancestral allele, has been associated with

a higher transcription rate. The presence of

this protein diminishes the cholesterol levels

in the HDL

, causing an increase in the risk

of developing cardiovascular diseases and,

therefore, increasing the risk of presenting

an accelerated aging process.

Some studies have demonstrated that

the antioxidant capacity of HDL and the

enzymatic activity of PON1 decreases with

age

. Likewise, the risk allele for the rs662

polymorphism in the PON1 gene has been

associated with the reduction of the enzyme

Relationships between genetic vascular risk polymorphism and aging in Venezuela 291

Vol. 64(3): 281 - 295, 2023

arylesterase activity, which negatively af-

fects the antioxidant capacity of HDL

. This

could explain the relationship found in this

study between the risk allele for the rs662

polymorphism and the accelerated process

of aging. In this line of thought, the risk al-

lele frequency has also been reported to de-

crease in nonagenarian and centennial indi-

viduals

Concerning the genes involved in car-

diovascular homeostasis, the allelic variant

655T of rs1801133 polymorphism in the

MTHFR gene has been linked to a decrease

in MTHFR enzymatic activity which in turn

causes an increase in blood homocysteine

being reported a decrease of up to 70% in

homozygotes individuals for the risk allele

. This homocysteine level change has been

associated with a decline in physical func-

tions. Proposed mechanisms regarding this

outcome include direct endothelial damage

caused by the generation of potent reac-

tive oxygen species not only in endothelial

tissues but also in proteins and DNA, along

with an increase in the amount of telomere

length loss

. The rs1801133 polymorphism

has also been related to longevity with a de-

creased allelic frequency in centenary indi-

viduals for the risk allele 655T

The allelic variant Del of rs4340 poly-

morphism in the ACE gene has been associ-

ated with an increase in plasmatic and cardi-

ac ACE activity, causing an overexposition to

high levels of Angiotensin II that on its own

has been linked to a diverse repertoire of

cardiovascular diseases such as hypertension

and myocardial infarction

. It is worth not-

ing that, while there has been a significant

quantity of studies concerning this polymor-

phism, the mechanism by which it operates

has not been elucidated completely; this due

to the intronic nature of this polymorphic

variant.

The allelic variant 803C of rs699 poly-

morphism in AGT gene has been reported to

increase plasmatic AGT protein production

up to 20% in homozygote individuals for the

risk allele

; this increase has been adjudi-

cated to a linkage disequilibrium between

rs699 and rs5051 polymorphisms the last

one located in the gene promoter imposing

an augment in transcriptional activity of the

AGT gene

. While this polymorphism has

been linked with cardiovascular homeostasis

alterations, few studies associate this variant

with accelerated aging, some postulating it

as a protection factor

46,47

. In contrast, others

suggest there is no relationship at all. Simi-

larly, to rs4340 polymorphism in ACE, the

increase in AGT transcription rate is deeply

linked to an increase in Angiotensin II lev-

els, promoting the risk of developing cardio-

vascular diseases. However, in this case, the

increased allelic frequency of the risk allele

reported in advanced-age individuals sug-

gest the existence of a protection element

conferred by this variant; this protection has

been postulated to exist due to the role as a

skeletal muscle growth factor of angiotensin

46--48

Lastly, the allelic variant 894T of

rs1799983 polymorphism in the NOS3 gene

has been associated in previous studies with

an increased probability of developing car-

diovascular events and preeclampsia due

to alterations in the NOS3 enzyme func-

tion

. In a previous case-control study we

measured the nitric oxide concentration in

serum through non-enzymatic colorimetric

assays reporting that individuals carrying

the 894T allelic variant showed a reduction

of 46.47% in nitric oxide serum levels when

compared with GG homozygote individuals.

These results were statistically significant

and showed that the presence of 894T can

contribute to an increased risk of develop-

ing hypertension of up to four times in TT

homozygote individuals (OR: 4.17; CI: 1.06-

19.11; p<0.05) when compared to GG ho-

mozygote individuals

In summary, according to the results of

the OR analyses, the polymorphic variants

considered in this study were not associated

with the development of accelerated aging

in a statistically significant way, except for

the rs4340 polymorphism in the ACE gene

292 Álvarez et al.

Investigación Clínica 64(3): 2023

(recessive model). These results could be

related to the sample size since it is essen-

tial to obtain statistically significant results.

Nevertheless, the multivariable analysis

showed a significant association between the

variable’s obesity, hypercholesterolemia and

hypertriglyceridemia, age, body mass index,

APOB rs693, and PON1 rs662 polymorphism

with the development of accelerated aging

in the G1 group. Also, the variables sex, hy-

pertriglyceridemia, smoking, age, body mass

index, systolic hypertension, and MTHFR

rs1801133 polymorphism were linked with

accelerated aging in the G2 group. Our find-

ings showed that genetic and environmental

factors are associated with an accelerated

aging process.

The knowledge of the genetic profile

is of great importance to complement the

biochemical and clinical information of the

individuals. The integral consideration of

these parameters will allow the application

of preventive antiaging medicine in an indi-

vidualized way by making nutritional recom-

mendations and modifications in lifestyle to

reduce the incidence of diseases typically

associated with age, such as cardiovascular

diseases, and promote healthy aging.

ACKNOWLEDGMENTS

The authors are immensely grateful to

Ph.D. José Bubis for his comments on earlier

versions of the manuscript, although any er-

rors are our own and should not tarnish the

reputation of this esteemed person.

Funding

This research was funded by FONACIT,

grant number G2005000398.

Competing interests

The authors have no relevant financial

or non-financial interests to disclose.

ORCID Numbers

• Carlos Álvarez (CA):

0000-0003-3773-2363

• Andrea Bullones (AB):

0000-0002-6765-8486

• María Angélica Medina (MAM):

0000-0001-8310-5008

• Anna Vargas (AV):

0000-0003-2658-369X

• Antonietta Porco (AA):

0000-0001-5134-1284

• Juan Carlos Méndez (JCM):

0000-0002-2425-6911

• Carolina Pestana (CC):

0000-0002-5590-5304

Author’s Contributions

CC direction and designed research.

CA, AB, MAM, AV and AA performed re-

search and analyzed data. CC, AP and AC

wrote the paper. JCM analyzed the clinical

data. All authors read and approved the final

manuscript.

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