Peripheral blood samples were taken in sterile EDTA tubes (Kemico Vacutainer, Egypt) for genotyping and hematological evaluations. Additionally, blood aliquots were taken in serum tubes for other laboratory tests. Sera were stored at –20°C until use. All samples were identified and given matched numbers that corresponded to all investigations.

Physical inspections and routine questionnaires were done for all patients. Hematological parameters, such as hemoglobin, white blood cells (WBCs), and platelets, were investigated using Sysmex KX21 Hematology Analyzer (Sysmex Corporation, Japan). Other standard biochemical analyses, such as fasting blood sugar, aspartate transaminase (AST), alanine transaminase (ALT), urea, and creatinine (Biosystem, Spain), were performed.

Single nucleotide polymorphisms (SNPs) under investigation were selected according to PubMed published data (SNP database). To see if the promoter region harboured any genetic variants susceptible to CHD, we chose two tagSNPs, 5’ upstream, of the TGFBR2 gene (rs6785358 and rs764522).

In an EDTA-containing tube, approximately 4 mL of venous blood samples were collected. Genomic DNA extraction was done from samples using the phenol-chloroform method in the presence of proteinase K digestion (Sigma-Aldrich, Germany). Using a UV spectrophotometer (Spectronic 1201, Milton Roy, USA), the absorbance at 260 nm was used for DNA concentration, however, the A₂₆₀/A₂₈₀ nm was used for purity determination.

To examine the allelic variance, the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was done. The primer sequences of the rs6785358 were 5’-GAACTGCAAACAAGAGAATGGAT-3’ (forward) and 5’-TTAGAATTCTACCCTAATGATTGTAAGG-3’ (reverse), however, the rs764522 primers were 5’-GAGTGAAAGAGCCCAGAACG-3’ (forward) and 5’-GGGCTAGGCATCTTCTTTCC-3’ (reverse) [13]. A total volume of 10 μL of PCR reaction containing 10 ng of DNA, 0.5 pmol of each primer, and 1 × PCR master mix was prepared. Using an GeneAmp^TM PCR System 9700 (Thermo Fisher, USA), the amplifications were accomplished. The reaction program proceeded as follows: one cycle for 5 min at 95°C, 30 cycles at 95°C for 30 s, 30 s for annealing at 61°C (rs6785358)/63°C (rs764522), and 30 s at 72°C.

A final extension cycle at 72°C for 10 min. The PCR products were digested using the restriction enzymes BsuRI and MvaI (Thermo Fisher, USA), for rs6785358 and rs764522, respectively [13].

Fisher’s exact χ² test was used for Hardy-Weinberg equilibrium assessment (HWE 14 program in SPSS). The SPSS for Windows version 13.0 (SPSS Inc, USA) was used for performing statistical analyses. The Chi-square (χ²) test was done for the analysis of allelic variation among studied subject groups. The relation was illustrated as odds ratio (OR) with 95% confidence interval (95% CI) of the risk. The level of statistical significance was set at 0.05 (two tails). Also, Binary logistic regression was done for the association between different variables and the disease status.

The characteristics of the cases (n = 75), and control (n = 100) groups are listed in Table 1. Regarding age or sex, the demographic results revealed a non-significant difference between the studied groups (P > 0.05). However, non-significant differences among studied groups regarding all laboratory investigations (P > 0.05) were noticed, except AST, hemoglobin, and platelets (P < 0.05).

The MvaI-digested PCR fragments were separated using the agarose gel electrophoresis method. The PCR amplicon was seen at 192 bp. For rs764522, the C allele was not digested and appeared as a single band at 192 bp. However, the G allele strand was digested into 41 bp and 151 bp fragments.

In Table 2, there are different models for testing the association of TGFBR2 rs764522 gene polymorphism with CHD. Herein, the G/G genotype frequency is much higher among cases compared to controls (21.3% vs. 0.0%). This might imply that the G/G genotype is a predisposing factor to the occurrence of CHD. A positive significance in the codominant model (C/C vs. C/G vs. G/G; OR = 0.43, 95% CI = 0.02–0.90, P < 0.0001) was noticed, as well as, in the dominant model (C/C vs. C/G + G/G) and recessive one (C/C + C/G vs. G/G; P < 0.0001).

The TGFBR2 (rs764522) gene polymorphism cross interaction with gender of cases and controls is displayed in Table 3. The genotype C/G frequency was noticed to be lower in male cases than in females and controls (OR = 0.24, 95% CI = 0.07–0.84).

In Table 4, the TGFBR2 (rs764522) gene polymorphic genotypes are revealed among cases of different types of CHDs compared to controls. The C/C genotype frequency in the VSD cases was much lower compared to controls (14% vs. 40%). On the other hand, the G/G genotype frequency was higher in ASD cases compared to controls (20.9% vs. 0.0%) with a significant statistical difference (P < 0.0001).

For rs6785358, the BsuRI-digested PCR products were resolved on agarose electrophoresis. The results revealed that the A allele remained intact as a single 176 bp band. However, the G allele strand was digested into 147 bp and 29 bp fragments.

The different models for testing the association of TGFBR2 (rs6785358) gene polymorphism with CHD are given in Table 5. The results revealed that the G/G genotype frequency is much higher among cases compared to controls (18.7% vs. 4.0%, respectively). This might imply that this genotype is a predisposing factor to the occurrence of CHDs. Moreover, a positive significance in the codominant model (A/A vs. A/G vs. G/G; OR = 0.20, 95% CI = 0.06–0.66, P = 0.0073) was noticed, as well as in the recessive model (A/A + A/G vs. G/G; OR = 0.19, 95% CI = 0.06–0.60, P = 0.0018).

The TGFBR2 (rs6785358) gene polymorphism cross interaction with gender of subjects is shown in Table 6. The results indicated that the genotype G/G frequency was noticed to be higher in male than female cases and controls (OR = 0.10, 95% CI = 0.01–0.88 and OR = 0.22, 95% CI = 0.05–0.94, respectively).

The TGFBR2 (rs6785358) gene polymorphic genotypes among cases of different types of CHD compared to controls revealed that the G/G allelic frequency in the VSD cases was much higher in cases compared to controls (25.6% vs. 4.0%) with a significant statistical difference (P = 0.002, Table 7).

The Hardy-Weinberg genetic equilibrium analysis for the TGFBR2 gene variant (rs764522) showed a negative equilibrium in both the studied subjects and control samples, with significant P-values of 0.0008 and 0.0001, respectively. This deviation is likely due to the very low frequency of the G/G genotype among the controls. However, in the cases group, a positive Hardy-Weinberg equilibrium (P = 0.11) was observed.

Regarding another gene variant (rs6785358) within TGFBR2, the analysis revealed a positive equilibrium in both the studied patients and controls, with P-values greater than or equal to 0.05, indicating no significant deviation from the equilibrium.

Regression modeling is a statistical method employed to examine the connections between variables. In the context of assessing the correlation between genetic markers and disease status, logistic regression is commonly used. The analysis involved evaluating the association of each SNP. In cases of binary responses, the outcomes of logistic regression analysis are succinctly presented in Table 8, encompassing significance levels, OR, and 95% CI. The findings indicate a noteworthy association between the GG genotype of rs6785358, as opposed to the CC genotype, and cardiovascular disease in children. Additionally, the CC genotype of rs764522 exhibited a significant association with the disease. Conversely, the variable of sex displayed a lack of significant association with the disease.

This study showed that SNP rs6785358 and rs764522 of TGFBR2 gene were associated with an elevated risk of CHD in the Egyptian population. In the future, the results offer an opportunity for the development of a novel early genetic detection of CHD risk.