Premature coronary artery disease (CAD) has been proven to have a multifactorial etiology and is most likely a mixture of genetic and environmental factors, being the myocardial infarction occurrence, the most prevalent factor affecting offspring before the age of 55 in men and 65 in women [37,38]. In the Framingham study, after 8 years of follow-up of male and female offspring with parental history of premature cardiovascular disease (CVD) (father < 55 years, mother < 65 years), CVD increased 75% with paternal and about 60% with maternal history of premature CVD. After 16 years of follow-up, the study also found that a family history of early CAD (age <50 years) conferred a 44% increased risk of CVD mortality [11,12,39,40].

In the present study, 48.7% of atherosclerotic coronary artery disease was found in young men, with a mean age of 39.7 years at clinical diagnostic. It was observed that the majority were sedentary individuals, with overweight and visceral obesity, with no family history of premature CAD and high levels of fasting glucose, glycated hemoglobin, and very low levels of HDL-cholesterol. In the coronary angiography performed, no differences were observed in the coronary disease involvement, with the number of single and multivessel arteries being similar. Likewise, there was no predominant involvement of any coronary territory.

The prevalence of male cases agrees with several studies that confirm that men are more affected by early CAD [41,42]. This fact can be related to hormonal protection that women have while they do not enter the climacteric phase, with estrogen being a potent atheroprotective as exerts many beneficial effects against arterial diseases, including vasodilatation, acceleration of healing in response to arterial injury, arterial collateral growth and protection [43,44]. It is well established that smoking causes thrombosis, promoting the development of an inflammatory and thrombogenic profile, associated with reduced levels of prostacyclin action and increased levels of nitric thrombosis, favoring endothelial vasospasm occurrence [45]. However, no association with smoking was found, since 57% of the CAD group were not smokers.

Several studies have shown that a family history of early coronary artery disease increases CAD risk in first-degree family members increasing susceptibility to atherosclerosis growth [12,38,39,46]. However, in our study, 53% of CAD patients had no family history of CAD before in first-degree family members which disagrees with [47], who evaluated 165 North American patients with acute myocardial infarction under the age of 45 years, where it was observed that 70% were smokers and 70% had a family history of early CAD. Moreover, a large international study (INTERHEART) found an increased risk of myocardial infarction (MI) of 2.36 (Odds ratio) if at least one parent had MI and 6.56 (OR) if both parents had MI before age 50 [48]. Furthermore, when a section is made for Latin America, the INTERHEART study shows that interventions aimed at lowering blood pressure, and modifying lipids could have a large impact on the risk of acute myocardial infarction among this population [49]. Probably, the mixed population in Brazil, its genetic background, ethnicity, and other environmental exposure may influence these differences between populations around the world.

Regarding the lipid profile, patients showed similarity to what is observed in most studies that describe low levels of HDL-cholesterol (HDL-C), where all our patients had HDL-C lower than 40 mg/dl, being the lipid alteration, most found in individuals with early CAD [50– 52]. These studies emphasize that for HDL levels below 35 mg/dl, there is an increased risk of CAD occurrence by at least 4 times, and conversely, an increase of 2 mg/dl of HDL- C confers a reduction in the risk of new events and lower incidence of percutaneous coronary interventions [53]. Moreover, the Framingham study and others that followed could show that HDL-C is an independent cardiovascular risk factor and that the increase of HDL-C of only 10 mg·L⁻¹ leads to a risk reduction of 2–3% [54–57]. Despite the existence of consistent epidemiological evidence that attributes the decrease in HDL-C to an important atherosclerotic risk factor, its exact role in CAD pathogenesis is unclear. [58–60] did not observe further reductions in coronary heart disease (CHD) risk with HDL-C values higher than 90 mg/dL in men and 75 mg/dL in women. However, several studies have shown that not only the severity of angiographic lesions but also the risk of cardiovascular events related to low concentrations of HDL-cholesterol are due to an ineffective mobilization of cholesterol from the arteries and peripheral tissues, characterizing an ineffective reverse cholesterol transport [61,62]. It is believed that, in this context, the presence of genetic alterations, characterized by single nucleotide polymorphisms (SNP), may influence the presence or absence of these risk factors [63,64]. Moreover, a significant difference was also observed between groups in the levels of glycated hemoglobin. The literature shows that an altered glycemic profile and thus insulin resistance, before the diagnosis of diabetes, generates an imbalance in glucose metabolism that generates chronic hyperglycemia, which in turn triggers oxidative stress and causes an inflammatory response that leads to cardiovascular damage [65,66]. It is important to emphasize that most of our CAD cases were unaware of any change in the glycemic profile prior to their diagnosis.

As for the extent of coronary involvement, several authors argue that multivessel coronary disease is uncommon in individuals with early CAD [67–69]. Although previous studies have reported that coronary involvement in young patients is mainly single-vessel and to a lesser extent, this study did not demonstrate a significant predominance of the pattern of single-vessel or multivessel coronary involvement. To date, we have not identified studies that correlate the presence of genotypic alterations with the predominance of involvement of specific coronary territories. Some studies [70–73] point to the possibility that patients with specific genotypic characteristics could present a greater severity in the extension of the coronary territory demonstrating that combined polymorphisms could be associated with the extension of coronary disease and more severity of the disease [74].

One of the hypotheses raised for the presence of genetic influence is on Paraoxonase 1 (PON1), the main constituent enzyme of HDL-C particles which is responsible for its antioxidant, anti-inflammatory, and reverse cholesterol transport properties, that could be enrolled in promoting changes in HDL-C concentration [75–77]. The genetic alterations would make the individual predisposed to develop atherosclerosis early even in the absence of high levels of LDL cholesterol and could intensify the role of other risk factors such as low levels of HDL cholesterol, high blood sugar, overweight, and abdominal obesity.

Few studies have been performed to analyze the association of the PON1 L55M polymorphism with coronary heart disease, and many of these have shown inconsistent results [13,28,78]. Blatter et al., [79] and Mackness and Mackness [80] found that the PON1 L55M polymorphism has a significant effect on its activity independently. Schmidt et al.,

[81] also observed an independent association of CAD with this polymorphism in Austrian subjects.

Garin et al., [82] investigated C108T PON1 polymorphism (promoter region) in patients with ischemic heart disease and their results showed that individuals with CC genotype had significantly higher levels of HDL cholesterol than heterozygotes. In contrast, no significant difference was observed in LDL and HDL levels for the PON1 L55M polymorphisms. In the present study, an LL genotype prevalence for L55M PON1 polymorphism was found to be 2.54 times higher in CAD cases than in healthy controls. This finding disagrees with Bounafaa et al., [83] who showed in a sample of 305 North African individuals that those with the PON1 55 MM genotype had a higher risk of coronary heart disease than those with the 55 LL genotype, showing once more that etinicity may play a role in CAD. Moreover, several contradictions are noticed in available studies. In a review by Litvinov et al. [84], it was reported that the antioxidant activity of PON1 decreases sequentially in MM > LM > LL genotypes, with wild-type homozygotes (LL) showing about half the activity of the paraoxonase enzyme in comparison to homozygotes mutated (MM). Allelic variation frequency among populations is enormous. The frequency of polymorphic M allele is higher in whites than in blacks. A lower frequency of the M allele has been reported in the Chinese population [85].

As for C108T polymorphism, there was no significant difference in the presence between CAD cases and healthy controls, observing a balance in allele frequency. CC genotype is responsible for the highest activity of PON1, while TT genotype is related to low gene expression and reduced serum activity of the enzyme [86]. Litvinov et al. [87], demonstrated an association between C108T polymorphism and CAD. Levieve and James [88], observed that CC genotype carriers were protected from CAD involvement at age younger than 60 years, but not in older individuals [32]. Also was no association of C108T polymorphism with CAD after adjustment with traditional risk factors in this study.

When CAD cases and healthy control genotypes were analyzed together as a haplotype, there was an imbalance in haplotypes or linkage imbalance that seemed to be significantly associated with disease risk. This observation is supported by Gupta et al.[32], who emphasize that haplotype determination is gaining more and more attention due to the finding that multiple associated SNPs have a greater potential to promote disease than when they are analyzed alone. Much information is lacking regarding PON1 haplotypes and the risk of CAD. Levieve and James [88], also showed a strong linkage disequilibrium between C108T and L55M polymorphisms among CAD carriers.

An interesting observation in the present study is that a significant difference in TSH values was found in cases with CT or TT genotype for C108T polymorphism. It should be noted that, to date, we are not aware of any studies that evaluated the relationship between activity or concentration of human PON 1 with thyroid function and its consequences in early CAD development.

Several studies recognize the relationship between higher TSH values, even within a normal reference value, and cardiovascular risk, but in general they correlate this finding with the fact that these TSH values are associated with a diagnosis of metabolic syndrome [89–91]. This observation may agree with the actions of thyroid hormones on the cardiovascular system. It is known that myocardium and vascular endothelial tissue have receptors for such hormones and are sensitive to changes in their serum concentrations, even in minimal variations [92]. These observations lead to the discussion of whether TSH serum values added to PON1 polymorphisms contribute to an environment of higher cardiovascular risk.

Finally, in this study, we observed through multinomial analysis by logistic regression that, regardless of the presence of PON1 polymorphisms evaluated here, the risk intensity of symptomatic coronary disease at an early age is 3 times greater in those individuals with high total cholesterol and about 9 times if they also have low levels of HDL cholesterol. The presence of the allelic mutation will indirectly interfere with disease developing risk by promoting a favorable environment such as overweight and visceral obesity that contribute to metabolic alterations occurring mainly in glycemic and lipid profile and of an atherogenic character in young patients. To the best of our knowledge, this is the first time that these genetic modifiers are described in young Brazilian subjects, being an important risk factor that can be used in practice clinics to manage patients with CAD.