Blood Samples

A total of 80 healthy volunteers, randomly chosen, were enrolled in this study, with an age range of 20-50 years. Individuals who suffered from any history of disease or conditions that affect the immune system, such as malnutrition, infections (HIV, CMV, measles, HBV, HCV, HAV, HEV, etc.), chronic diseases (hepatic, renal, pulmonary, etc.), malignancies, surgery, trauma, chemotherapy, wounds, any medication influencing the immune system, infections of upper or lower respiratory tract, gastrointestinal tract or urinary tract infections, were excluded. The blood samples were collected from the individuals in Al-Imam Al-Sadiq hospital in Babylon city in Iraq, an ethical consent was signed by each volunteer. Two milliliters of the venous blood sample were obtained from each participant in a gel tube, and the blood was drawn by disposable syringes in a sterile condition. The collected blood was centrifuged to separate serum to be used later for TNF-α, IFN-γ and TGF-β assessment.

DNA extraction and molecular detection

The DNA was extracted from the blood samples by using the gSYNC™ DNA Extraction Kit (Geneaid Company) The primers used in this study were designed by Primer3plus bioinformatics tool (https://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi). Conventional PCR was used to amplify the DNA fragments that contain the targeted sequence (393 bp) of the ZEB2 gene located on the long arm of chromosome 2. A total volume of 25 μl was used in the PCR reaction (3 µl DNA, 1µl from each primer, 12.5 µl master mix, and 7.5 µl nuclease free water). The PCR program used to amplify the target sequence consisted of 35 cycles, each cycle included denaturation for 30 seconds at 94ºC, annealing for 30 seconds at 59ºC and extension for 30 seconds at 72ºC . Agarose gel electrophoresis was used to separate PCR product bands on 2% agarose gel stained by fluorescent Redsafe dye. The gel electrophoresis system was set at 70 volts for 60 minutes, then the gel was displayed under UV transilluminator.

Nucleotides sequencing and analysis

All the 80 PCR product samples were sent to the Macrogen public biotechnology corporation, South Korea, to perform nucleotide sequencing. Sanger sequencing method was applied using an automated DNA sequencer. The results of sequencing were received by email, analyzed manually by using bioinformatics tools and aligned with human reference gene sequences already deposited on the National Center for Biotechnology Information (NCBI). The alignments were done by using the Basic Local Alignment Search Tool (BLAST) provided by NCBI. The sequenced region of the target gene was analyzed by using Molecular Evolutionary Genetics Analysis X (MEGAX), in which the multiple alignments of the sequences were performed by the CLUSTALW program to confirm the presence of variations detected by the BLAST tool [14]. Locations of all variants detected in the current study were reported and checked to identify the type of variants and predict their functional consequences by using tools provided by Ensemble Genome Browser. In addition, the aforementioned browser was also used to identify presence of known, unknown, and novel variants in the current study.

Enzyme Linked Immunosorbent Assay (ELISA)

The ELISA kits (Elabscience company) were used in this study to determine the serum levels of human TNF-α, TGF-β and IFN-γ.

Statistical analysis

To determine the effect of different groups on the study parameters, the Statistical Analysis System (SAS, 2012) was utilized. To make comparison between means, the Least Significant Difference –LSD test (ANOVA) was performed. The Chi-square test was employed to make comparisons between percentages. 

The age of included 80 individuals ranged from 20-50 years old. Of the participants 46% were between 20–29 years old, 38 % were between 30–39 years old, and 16% were 40–49 years old. Apparently healthy individuals aged 20 to 50 years old were selected randomly to be enrolled in this study because they had an optimal immune system compared to individuals over 50 years old. The sample mean± SD for age was 31.2±6.87 years (ranged 20-48 years). The number of males was 60 (75%), and the number of females was 20 (25%) among the individuals. Results of the TNF-α serum level showed that its mean was 36.575, the standard deviation recorded 21.56, the median was 32.85, the standard error was 2.422, the minimum and maximum values were 7.7 pg/ml and 96.4 pg/ml, respectively. While the normal range value of TNF-α was <7.82 pg /ml (Table 2). The IFN-γ level revealed a mean of 9.46, the standard deviation recorded 4.69, the median was 9.106, the minimum and maximum values were 2.13 pg/ml, and 24.38 pg/ml, respectively. The standard error was 0.058, while the normal range value of IFN-γ was <15.63 pg/ml (Table 2). The findings also showed that the mean of TGF-β was 1482.68, the standard deviation recorded 505.76, the median was 1433, the minimum and maximum values were 101 and 2353, respectively, and the standard error was 56.82. While normal range values of TGF-β was 256.64 -1334 (Table 2).

Conventional PCR was used to amplify the DNA target region in the ZEB2 gene, including 5`UTR region (166 bp) and exon 1 region (181 bp). The PCR product (393 bp) was detected in all of the study samples, this indicates the presence of the target region in the ZEB2 gene. The genetic analysis of the 80 samples showed that 34 samples were identical to the reference gene sequence without any variations detected. While the other 46 samples showed different types of variations in the ZEB2 gene. The number of variants present in the samples was different from one sample to another, 23 out of 46 samples showed single variation in their sequences, seven samples showed double variations, the remaining 16 samples showed multiple variations. According to the effect of the ZEB2 gene variations on TNF-α, TGF-β, and IFN-γ serum levels, the results revealed a significant difference between Identical (ID) group (42.5%) (Identical: no variations were detected), single variation group (28.75%) (One variation was detected), double variation group (8.75%) (Two variations were detected) and multiple variation group (20%) (More than two variations were detected)  in both IFN-γ and TGF-β with a p-value of (0.0518) and (0.0368), respectively. While the results displayed non-significant differences between the same groups in the level of TNF-α (p value= 0.703) (Table 3).

The current study also showed an increase in the serum level of TGF-β with an increase in the number of variations in the ZEB2 gene (double and multiple variation groups). These results were similar to the ones which were reported by Li et al., (2017) [14] who found that the mutation and deletion in the ZEB2 gene can cause overexpression in TGF-β and demonstrated that the ZEB2 gene inactivation within the HSPCs (hematopoietic stem/progenitor cells) correlates with modifications in expression of genes involved in cytokine receptor signaling. These alterations might affect the differentiation of numerous hematopoietic lineages by modulating signal transmission. In this study, a positive relationship between TNF-α and TGF- β was found in volunteers who had multiple genetic variants, as they had a higher rate than volunteers with identical genetic sequences.

While in the case of IFN-γ, the relationship was inverse, the results showed that identical genetic volunteers had a higher mean value than volunteers who had multiple genetic mutations.