The Association between Single Nucleotide Polymorphisms rs1042522 and rs1642785 in the TP53 gene and Acute Myeloid leukemia in a sample of the Baghdad/ Iraq population

Acute myeloid leukemia (AML) represents the most prevalent type of acute leukemia in adults and is responsible for approximately 80% of all cases .The tumor suppressor gene ( TP53 ) is a gene that has been frequently studied in cancer

Tumor protein 53 (TP53 gene) is found on the short arm of chromosome 17 (17p13). TP53 gene encodes the p53 protein, which has tumor suppression as its main biological function 5 . The mutation rate of TP53 in all human cancers is between 25% and 40%; therefore, it is one of the most frequently mutated genes in human cancers 6 . TP53 is essential for regulating the tumor cell cycle, DNA repair, and apoptosis. The stimulation of TP53-dependent pathways by external or internal cellular stress signals influences metastasis, progression, cancer cell genesis, and preventing the growth of damaged cells with oncogenic ability. Furthermore, TP53, as a transcription factor, can activate a wide range of genes to promote these tumor suppressing processes 7 . Genetic studies employing polymorphisms that can assist in demonstrating associations between some molecules and diseases. The TP53 gene contains high-frequency, functional single-nucleotide polymorphisms (SNPs) that can change the function of the p53 protein 8 . The TP53 gene has more than 200 single nucleotide polymorphisms (SNPs). In TP53 gene, several SNPs have been investigated and found to be associated with the high risk of several types of human cancer 9 . One of the most widely studied polymorphisms in TP53 gene is rs1042522, which converts the amino acid at codon 72 from arginine to proline. The substitution of this amino acid affects the structure and function of the p53 protein by causing structural modifications 10 . The rs1042522 SNP was investigated for its role in disease risk for the vast majority of common types of cancer, including prostate, breast, and lung cancers [11][12][13] . Another SNP, rs1642785, is found on intron 2 of the TP53 gene and is associated with an increased cancer risk 14 . This study aims to see if there is a relationship between two TP53 polymorphisms (rs1042522 and rs1642785) and the risk of evolving acute myeloid leukemia in some Iraqi patients.

Subjects
This study has been designed and performed according to the institutional bioethical guidelines established by the Ethical Committee in the College of Science/University of Baghdad. Sixty patients (26 males and 34 females) and sixty controls (26 males and 34 females) have been matched in gender and age, all with an age range of 15-65. From February 2021 to August 2021, patients were chosen from the Baghdad teaching hospital in the medical city.

Blood Samples
About 3 ml of venous blood from all patients and the control group have been collected by vein puncture using a 5 ml syringe. A volume of 2 ml has been transferred into an EDTA tube and then stored at 4 °C until the extraction of DNA has been carried out.

Genomic Genotyping of TP53 rs1042522 and rs1642785 SNPs
The human TP53 gene is located on chromosome 17 and is composed of 10 introns and 11 exons. TP53 rs1042522 is located on chromosome 17:7676154bp in exon 4. TP53 rs1642785 is located on chromosome 17:7676483bp in intron 2. Genomic DNA has been extracted from the whole frozen blood that was collected in EDTA tube by using the commercial Easy Pure® blood Genomic kit followed by concentration and purity measurements. TP53 rs1042522 and rs1642785 SNP primers have been designed using Beacon Designer 8 and provided by Bioneer Company (Korea). Table 1 shows the forward and reverse sequences of these primers. Real time PCR amplification has been carried out with a total volume of 20µl, including the following: the master mix 10μl, forward and reverse primers 1μl each, DNA template 3µl and nuclease free water 5µl. For rs1042522, the real-time PCR conditions have been as follows: one cycle of initial denaturation at 94 °C for 1 minute, followed by 40 cycles of denaturation at 94 °C for 5 seconds, annealing at 52 °C for 15 seconds, extension at 72 °C for 20 seconds, and one cycle of HRM analysis at 60 °C to 95 °C for 2 seconds. And as follows for rs1642785: one cycle of initial denaturation at 94 °C for 1 minute, followed by 40 cycles of denaturation at 94 °C for 5 seconds, annealing at 56 °C for 15 seconds, extension at 72 °C for 20 seconds, and one cycle of HRM analysis at 60-95 °C for 2 seconds.

Results and Discussion
The results of nanodrop indicated that the genomic DNA was variable in concentration as it ranged from 7-55ng/µl. The purity ranged from 1.78-1.9 these results depended on white blood cells count and blood sample freshness.

Genotyping of TP53 gene by using HRM Real Time PCR
The closed tube technique called high-resolution melting (HRM) analysis, was used to evaluate an intercalating dye's fluorescence reduction in a double-strand DNA dissociation process. Because of its simplicity, adaptability, nondestructive nature, exceptional sensitivity, and precision, this technique has gained popularity over the past decade. The HRM analysis gives sequence-associated melting profiles and can show single-nucleotide-level changes in genotype 17 .

Detection of the TP53 gene (G>A rs1042522) by using HRM Real Time PCR
The SNP TP53 (G>A rs1042522) located on chromosome 17 (exon 4) has been found in three genotypes GA, AA, and GG and two alleles G and A in both control and patient subjects. Melt and fluoresce difference curves have been obtained by HRM assays for the rs1042522 SNP of the TP53 gene that has a basic relationship with AML. Figs. 1. A and B, Shows the output curve of HRM for heterozygous, homozygous and wild type rs1042522 SNP of the TP53 gene by real time PCR depending on melting temperature. Hardy-Weinberg equilibrium (HWE) analysis in the control group has shown that genotypes were not compatible with equilibrium and there was significant differences p = 0.003 have appeared between the expected and observed genotype in the control group that due to small size of sample, whereas the variation in the AML patient group was compatible and non-significant difference with p = 0.086 as shown in Table 2. The genotypes distribution of the G>A rs1042522 SNP in the patient group was 26.66%, 60% and 13.33% for homozygous wild type, heterozygous, and another homozygous variant, respectively, while genotypes in the control group were 70%, 20%, and 10% for homozygous wild type, heterozygous, and homozygous variant, respectively. Inspecting TP53 gene genotypes and allele frequencies in the control and patient groups revealed that there was significant variation between these frequencies, with an increase in the A alleles 43.33% vs. 20% and a decrease in the frequencies of the G allele 56.67% vs. 80. As shown in Table 3, the GA genotype represents risk factor (OR, 7.8; CI, 3.2-18.4; p=0.0001), the AA genotype represents risk factor (OR, 3.5; CI, 1.1-10.2; p=0.04), and the allele A represent risk factor (OR, 3.0; CI, 1.7-5.4; p=0.0001). In this study, it has been demonstrated, according to qPCR results, that the rs1042522 SNP was found in the examined sample; therefore, this SNP can be considered a risk factor for acute myeloid leukemia, which is consistent with the findings of other researchers 18 . Who assert a connection between the risk of AML and rs1042522. The main function of the TP53 tumor suppressor gene is to protect the genome from destruction. Polymorphisms have become an interesting topic in cancer studies 19 . The rs1042522 SNP is a common TP53 SNP that has been found on codon 72 and encodes for two alleles with different functional properties via transversion mutation 20 . TP53 Pro72Arg (rs1042522), located in proline rich domain of p53, is essential in the normal function of p53. The variant allele of rs1042522 has been reported to decrease apoptotic activity. Studies demonstrated that the arginine (Arg) variant is able to induce apoptosis faster and more efficiently than proline (Pro), while the Pro variant is better for inducing cycle arrest due to the fact that the Pro amino acid of p53 protein is weaker for apoptosis induction and also for suppressing cellular transformation compared to Arg amino acid. It has been reported that rs1042522 in TP53 gene can increase risk of cancers (including leukemia), and be associated with poor overall survival (OS) in patients with AML. As a result, the TP53 variant genotypes (rs1042522) could theoretically be risk factors for cancer progression and/or development 21,22 . The distribution of TP53 (rs1042522) genotypes in Iraqi patients with acute myeloid leukemia and control groups was studied, and the results show that this SNP plays a vital role in the progression of AML.

Detection of the TP53 gene (G>A rs1642785) by using HRM Real Time PCR
The SNP TP53 G>A rs1642785 located on chromosome 17 (intron 2) has been found in three genotypes GA, AA, and GG and two alleles G and A in both control and patient subjects. Melt and fluoresce difference curves have been obtained by HRM assays for the rs1642785 SNP of the TP53 gene that has a basic relationship with AML. Fig.2. A and B Shows the output curve of HRM for heterozygous, homozygous and wild type for rs1642785 SNP of TP53 gene by real time PCR depending on melting temperature. Hardy-Weinberg equilibrium (HWE) analysis in the control and patient groups has shown that genotypes were compatible with equilibrium and that non-significant differences p = 0.087 have appeared between the observed and expected genotype in the control group, but the variation in the AML patient group was non-significant with p = 0.40 as shown in Table 4. The genotypic distribution of the (G>A rs1642785) SNP in the patient group was 36.66% homozygous wild type, 43.33% heterozygous, and 20% rare homozygous variant, while genotypes in the control group were 70%, 23.33%, and 6.66% for homozygous wild type, heterozygous, and homozygous variant, respectively. Inspecting TP53 gene genotypes and allele frequencies in the control and patient groups revealed that there was significant variation between these frequencies. The results showed an increase in A allele frequency 41.67% vs. 18.33% and a decrease in G allele frequencies 58.33% vs. 81.67%. As shown in Table 5, the GA genotype represents risk factor (OR, 3.5; CI, 1.5-8.12; p=0.002), the AA genotype represents risk factor (OR, 5.7; CI, 1.6-19.5; p=0.005), and the allele A represent risk factor (OR, 3.1; CI, 1.7-5.7; p=0.0001). In this study, it has been demonstrated, according to qPCR results that the rs1642785 SNP was found in the examined sample; therefore, this SNP can be considered a risk factor for acute myeloid leukemia, which is consistent with the findings of other researchers 23 , who indicated that there was an association between rs1642785 and AML risk. Several studies investigated the relationship between rs1642785 genotypes and cancer. SNP rs1642785 is found on intron-2 of the TP53 gene. Introns are often involved in the regulation of gene expression and DNA-protein interactions and mutations in intron sequences may affect these functions. Therefore, TP53 mutations in the intronic region may initiate aberrant premessenger mRNA splicing, producing an mRNA that may be translated into defective p53 protein, which increases the likelihood of a deleterious phenotype that inhibits the apoptotic pathway and prolongs cell survival [24][25][26] . The distribution of TP53 rs1642785 genotypes in Iraqi patients with acute myeloid leukemia and control groups was studied, and the results show that this SNP plays a vital role in the progression of AML.

Conclusion
In conclusion, this case-control pioneer study showed that there was an association between the SNPs (rs1042522 and rs1642785) in the TP53 gene (exon 4, intron 2) with AML, which could be risk factors for AML in the Iraqi population.