Mutations in the TP53, VEGFA, and CTH Genes as Key Molecular Markers for the Diagnosis of Glioblastoma

Background Brain cancer, particularly glioblastoma (GBM), is a global health problem. Despite therapy advances, GBM patients have a poor prognosis. The progression and etiology of GBM may be linked to gene polymorphisms in the VEGFA, TP53, and CTH genes, among others. However, the genetic variations and their interaction in GBM are not fully understood. This study examines the effects of mutations in the VEGFA, TP53, and CTH genes on GBM. Methodology Tissue and blood samples were obtained for hematological, biochemical, and genetic analysis from 18 patients diagnosed with GBM as well as 28 healthy individuals. Standard methods were adopted to perform hematological and biochemical analyses, whereas mutational landscape and expression profiles were obtained from publicly accessible databases. Tissue samples were processed for genomic DNA extraction, and genotype determination was carried out through conventional polymerase chain reaction (PCR) and Sanger sequencing. Results The study involved 18 patients with grade IV GBM before treatment and 28 healthy individuals. The patients consisted of 11 men (61%) and seven females (39%), while healthy individuals included 14 (50%) males and 14 (50%) females. Sixty-seven percent of patients were under 50, 17% between 51 and 60, and 17% over 61, compared to healthy individuals who were 61% under 50, 7% between 51 and 60, and 32% over 60. GBM patients showed higher neutrophil and monocyte counts (median 81% (63.9, 83.5) and 4.2% (3.8-7.3)), respectively, and lower lymphocyte counts (median 13.4% (8.8, 28.40)) compared to controls. The median values of aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP) showed no significant differences between the control and GBM groups. GBM patients had significantly higher median CRP levels of 2.55 (1.6, 98) than controls. Analysis of databases revealed a high prevalence of mutations in TP53, with splice region variants, missense variants, and intron variants being the most common. VEGFA and CTH also displayed mutations, primarily missense and intron variants. Gene expression analysis showed significantly higher levels of TP53 and VEGFA in GBM patients compared to controls. CTH expression also exhibited a slight increase in GBM patients. Sanger sequencing identified three mutations in the TP53 gene, including a novel mutation (11915C>A) not previously reported in external databases. Additionally, novel mutations were found in the VEGFA (841G>GA, 919T>TG) and CTH (28398A>AC, 28399A>AT) genes. Conclusions This study highlights the immune dysregulation, inflammation, and genetic variations in GBM. The findings emphasize the potential importance of the TP53, VEGFA, and CTH genes as targets for therapies and diagnostic biomarkers of GBM. Further study is necessary to comprehend these genetic variations' functional implications and their use in personalized GBM treatment.


Introduction
Glioblastoma (GBM) is the most common and deadly primary CNS malignancy.Brain tumors and nervous system malignancies will account for 300,000 new cases and 250,000 deaths a year, 48% of CNS tumors are GBM [1].In 2021, the WHO classified brain tumors according to genetic changes and histologic characteristics.The four types of GBM are adult-type diffuse gliomas, pediatric-type diffuse low-grade gliomas, pediatric-type diffuse high-grade gliomas, and confined astrocytic gliomas [2].
Overexpression of vascular endothelial growth factor (VEGF) is a feature of GBM.VEGF stimulates extensive angiogenesis and has a direct correlation with the malignancy and prognosis of GBM [3].Accordingly, single nucleotide polymorphisms of this gene could play an important role in structural and functional alterations, leading to overexpression of this gene in GBM [4].The mutant variant of TP53 promotes tumor cell proliferation and accelerates the malignant transformation of astrocytic tumors [5].This variation is often connected with the IDH-1 mutation (65%-90% of cases), but the TP53 gene mutation occurs in only 30% of IDH wild-type GBM [6].
Hydrogen sulfide (H 2 S) is widely recognized as a carcinogenic gas and has the potential to be a target for cancer therapy and detection due to its role in cancer progression.Some enzymes, like cystathionine βsynthase (CBS) and CTH, need pyridoxal 5' phosphate to do their job.These enzymes are responsible for the conversion of L-cysteine into endogenous H 2 S inside human tissues.The liver, kidney, and brain contain In this study, we selected three commonly studied variations in the TP53, VEGFA, and CTH genes.The purified DNA was amplified individually for each genetic variant using conventional PCR on a Veriti 96thermal cycler (Thermo Fisher Scientific, Waltham, USA) using the following primers: TP53 forward, 5′-TCCCCCTTGCCGTCCCA-3′ and reverse, 5′-CTGGTGCAGGGGCCACGC-3′; VEGFA forward, 5′-CTCGGTGCTGGAATT TGATATTC-3′ and reverse, 5′-CAAAAGCAGGTCACTCACTTTGC-3′; CTH forward, 5′-GGACTTCTTGAGGAGTTGAAGC-3′ and reverse, 5′-ATTCTCACCTCCTTCAGAGGC-3′.
Initially, a ready-to-use master mix (ADDBIO INC., Daejeon, Korea) was used, containing Taq DNA polymerase, dNTPs, KCl, and reaction buffer.The following thermocycling conditions have been used for PCR: initial denaturation at 95°C for 5 min, followed by 35 cycles of 95°C for 30 sec; different annealing temperatures were used for each polymorphism (TP53 at 60°C, VEGFA at 55°C, CTH at 54°C) for 30 sec; and elongation at 72°C for 1 min, followed by a final extension step at 72°C for 5 min.The PCR products for TP53 and CTH were separated by 2% agarose gel electrophoresis.VEGFA was separated on 3% agarose gel electrophoresis, compared with the 100-bp DNA marker (GDSBio, China), and stained with a safe dye (DSView Nucleic Acid Stain, GDSBio) before being cast into the tray and visualized using a gel documentation system (UV transilluminator UVP, UK).Following the PCR procedure, the products were sent for sequencing using the same forward primers for each particular polymorphism using an automatic ABI PRISM 3130 DNA sequencer (Applied Biosystems, USA).Analysis of Sanger sequencing data was achieved using the Mutation Surveyor software (Soft Genetics, State College, USA), and the mutation result was compared with public databases, including gnomAD, dbSNP, ClinVar, and COSMIC.

Statistical analysis
For biochemical and hematological analysis, comparisons between patients with GBM and healthy individuals were performed using a Mann-Whitney test, and values were presented as medians and 25% and 75% quartiles.Whereas for gene expression retrieved from the GENT2 database, comparisons between patients with GBM and healthy individuals were performed using an unpaired t-test, and values were presented as mean±SEM.All data were subjected to normality testing, including the D'Agostino and Pearson omnibus, Shapiro-Wilk, and KS tests.The graphics, computations, and statistical analyses were generated using GraphPad Prism software (version 10; GraphPad Software, Inc.).A p-value of <0.05 was considered statistically significant.

Ethical considerations
The Scientific Research Division's Research Ethics Committee of the University of Duhok approved the study protocol under approval number (15092021-9-13), and all procedures contributing to this work met national and institutional human experimentation committees' ethical standards and the Declaration of Helsinki.

Patient characteristics
This study includes a cohort of 18 GBM patients and 28 healthy individuals.Among these patients, 11 (61.11%) were men and seven (38.88%) were women.Individuals who participated were categorized based on their age, with 12 (66.66%)being under the age of 50, three (16.66%)being between the ages of 51 and 60, and three (16.66%)being older than 61, while healthy individuals were 14 (50%) male and 14 (50%) female, with 17 (60.71)being under the age of 50, two (7.14%) being between the ages of 51 and 60, and nine (32.14%) being older than 61.All patients included in this study were diagnosed with GBM tumors, specifically grade IV.

Mutation and expression retrieval from databases
The distribution of mutations within these genes retrieved from the gnomAD database, including various classes of mutations, is briefly presented in Table 1.A total of 2041 mutations were identified in the TP53 gene, comprising significant mutations in the splice region variant (70), missense variant (564), and intron variant (902).VEGFA showed a total of 1862 mutations, with missense variant (593), intron variant (772), and synonymous variant (246) being the most prevalent.A considerable percentage of the 1839 mutations identified in CTH were intron variant (920), missense variant (509), and splice region variant (52).A summary of the mutations retrieved from the ICGC database is presented in Table 2 and Table 5 in Appendices, which classify them according to their clinical significance and impact.A total of 1079 highimpact mutations were identified in TP53, with 136 of them being classified as clinically significant.The majority of these mutations were composed of single-base substitutions.Among them were 25 missense mutations, one stop gained mutation, and nine likely pathogenic mutations, in addition to various pathogenic classifications: nine pathogenic, eight pathogenic/likely pathogenic, and nine likely pathogenic.Twenty-two high-impact mutations were observed in VEGFA, two of which were stop-gain mutations.Three of the 84 high-impact mutations identified in CTH were missense mutations.Notably, in this dataset, no clinically significant mutations were identified for VEGFA and CTH genes.The types and frequencies of COSMIC database mutations are summarized in Table 3 and Table 6 in Appendices.TP53 had a total of 773 mutations, with the majority being substitution missense mutations (515), deletion frameshift mutations (67), and substitution nonsense mutations (46).VEGFA showed a lower number of mutations, including 12 mutations, consisting of five substitution missense mutations and three substitution silent mutations.CTH had the lowest mutation number with a total of four mutations, mostly comprising two substitution missense mutations and two unknown mutations.Notably, the data we collected did not contain complex frameshift or whole gene deletion mutations in VEGFA or CTH.

Genes
TABLE 3: Summary of the mutations in the TP53, VEGFA, and CTH genes in GBM retrieved from the COSMIC database.

Mutation analysis
In the present study, we elected to genotype three different single nucleotide polymorphisms in the TP53, VEGFA, and CTH genes.Following DNA sequencing, we used a Mutation Surveyor to identify mutations in the TP53, VEGFA, and CTH genes (Table 4).The most nucleotide substitution that occurred in the TP53 gene was nine times C→G.The variant mutation (11897C>G) on chromosome position (17:7579472) has been previously described in external public databases, and its variant percentage in the present study was 100%.However, the C→A substitution in the mutation variants (11915C>A) on chromosome position (17:7579454) has not been previously described in the external databases, also its variant percentage was 100%.While, the C→CT substitution in the mutation variants (11846C>CT) also occurred on chromosome position (17:7579548), causing a change in amino acid Serine to Isoleucine, in which this mutation was not found in the external databases, and its variant percentage was 12.5%.

GBM: glioblastoma
In the VEGFA gene, heterozygous substitution and one homozygous substitution in the mutation variants (905C>CG and 905C>G) on chromosome position (6:43738350) has occurred in GBM patients, and this mutation has been previously described; its variant percentage was 37.5%; while, the G→GA substitution with mutation variant (841G>GA) that occurred on chromosome position (6:43738173), in which this mutation was not found previously in the databases, and its variant percentage was 12.5%.However, the T→TG substitution in the mutation variants (919T>TG) on chromosome position (6:43738364), in which this mutation was not found previously in the databases, and its variant percentage was 12.5%.
A total of seven mutations were identified on chromosome 1 at various positions of the CTH gene.Three of the newly found mutant variants in the CTH gene are associated with a change in amino acid level.The T→TC heterozygous substitution with mutation variant (28376T>TC) on chromosome position (1:70904776).However, four mutation variants (28400G>GT, 28495A>A, 28577T>T, and 28611C>C) on chromosome position (1:70904800, 1:70904895, 1:70904977 and 1:70905011) no amino acid changes were detected in any of them except the variants (28400G>GT,403S>S).Two mutation variants (28398A>AC and 28399A>AT) on chromosome position (1:70904798 and 1:70904799) caused a change in amino acid, nor were any of them reported in external databases, and its variant percentage for all mutations was 25%.

Discussion
This study examined a group of 18 patients diagnosed with GBM, an extremely severe form of brain cancer.
The patient group included more men than women, which is consistent with the reported higher incidence of GBM in men [11].The majority of individuals in both the patient (66.66%) and control (60.71%) groups were under 50 years old, with fewer falling into the 51-60 and above 61 age categories.This might be attributed to the rarity of GBM in younger individuals, along with the increased death rate related to the disease in elderly people [12].
Comparing the blood parameters of GBM patients to those of a control group shows that the development of GBM is linked to significant changes in immune cell profiles and signs of inflammation.GBM patients significantly increased their neutrophil levels, indicating the presence of a pro-inflammatory condition in the tumor microenvironment [13].In contrast, GBM patients had substantially lower lymphocyte numbers, indicating the presence of immune suppression or evasion mechanisms used by the tumor [14].Furthermore, the number of monocytes was considerably greater in individuals with GBM, which may indicate the presence of tumor-associated macrophages infiltrating the tumor [15].In addition, the GBM immunological microenvironment is inhabited by myeloid cells, including tumor-associated macrophages, myeloid-derived suppressor cells, neutrophils, and dendritic cells, indicating an increase in myeloid progenitors in GBM [16].Moreover, the markedly elevated levels of CRP in individuals with GBM indicate the presence of systemic inflammation associated with the tumor [17].Although there were changes in the immune system, the liver function markers AST, ALT, and ALP did not show any statistically significant changes between GBM patients and controls.Sarganas et al. [18] showed a substantial increase in liver function markers after temozolomide therapy.These results emphasize the complicated connection between immune dysregulation and inflammatory processes in GBM and highlight the potential diagnostic and therapeutic significance of monitoring immunological and inflammatory markers in GBM patients.
An in-depth examination of mutations retrieved from multiple databases provides essential information on the molecular basis of cancer, with a specific emphasis on essential genes, including TP53, VEGFA, and CTH genes.The gnomAD database revealed significant variations in the distribution of mutations across these genes, emphasizing the broad spectrum of mutation types and their incidences.An extensive number of mutations, such as splice region variants, missense variants, and intron variants, were found in the TP53 gene, indicating the complicated sequence of changes in this gene responsible for suppressing tumor growth.Variations in missense and intron mutations were frequently observed in the VEGFA and CTH genes, showcasing a diverse array of mutation patterns.The ICGC database uncovered several significant mutations in the TP53 gene, highlighting its importance in cancer development.In contrast, the VEGFA and CTH genes exhibited fewer variations, but there were noticeable differences in the kinds of mutations.Notably, there were no significant mutations found for the VEGFA and CTH genes in this dataset that had clinical significance.TP53 mutations were very widespread in the COSMIC database, exhibiting a range of mutation types such as substitution missense and deletion frameshift mutations.This indicates that the gene TP53 displays genetic heterogeneity in cancer.VEGFA and CTH genes had a lower number of alterations in comparison to TP53, primarily consisting of missense mutations.The current set of VEGFA and CTH genes did not contain any complex frameshift or whole gene deletion mutations.In general, these results provide significant knowledge about the genetic mutations in cancer-related genes and could direct future studies on targeted therapies and diagnostic approaches.
The analysis of genetic alterations from databases provided a roadmap for targeted sequencing in the second part of the study.The databases indicated the TP53 gene as a critical gene with frequent mutations, which led to further analysis in GBM patients.In these individuals, TP53 mutations, including potentially new ones, were actually found.Similarly, databases have shown possible roles for the VEGFA and CTH genes, but their changes seem to be less significant.Therefore, the current study examined the occurrence of mutations in TP53, VEGFA, and CTH, genes that are known to be associated with the development of GBM.The study identified numerous mutations in all three genes, providing valuable information on the possible genetic characteristics of GBM in the population under consideration.
The TP53 gene, which acts as a tumor suppressor, plays a crucial role in preserving the integrity of the genome and inhibiting excessive cell proliferation [19].Several types of malignancies, including GBM, frequently exhibit TP53 mutations [20].The investigation of the TP53 gene in GBM patients provided curious findings regarding specific mutations and their frequency.In total, three mutations in the TP53 gene of GBM patients were identified in three variants at various positions on chromosome 17.A noteworthy finding was the high frequency of C→G nucleotide substitutions in the TP53 gene, namely, the variation 11897C>G (chromosome 17:7579472), which was present in all GBM cases.This specific mutation has been recorded in external databases, confirming prior findings of its connection with the development of GBM [21].The 11897C>G mutation in our study cohort highlights its potential importance in GBM development.In addition, we found two previously unidentified mutations in the TP53 gene: 11915C>A, which is present in 100% of cases, and 11846C>CT, which has a frequency of 12.5%.External databases have not reported the 11915C>A mutation, suggesting the possibility of discovering a new variation.Similarly, the mutation 11846C>CT, while less common, results in an alteration of the amino acid sequence from Serine to isoleucine.The functional implications of these new mutations are still unclear.More studies are needed to determine their impact on the structure and function of the p53 protein, as well as their possible role in the development of GBM.
VEGF plays a crucial role in angiogenesis, and the growth and progression of various malignancies, including GBM, are associated with elevated VEGFA expression [4].The most frequently detected mutation was a hybrid of heterozygous and homozygous substitutions at locations 905C>CG and 905C>G on chromosome 6:43738350.We observed this mutation in colorectal cancer in our previous research but not in GMB [22].Mutations in this specific location of the VEGFA gene have the potential to affect the signaling pathways and stimulate the growth of blood vessels in the GBM.The observed prevalence of 37.5% indicates that this mutation could have significant consequences for a significant number of GBM patients.We have also identified two new mutations in the VEGFA gene: 841G>GA (chromosome 6: 43738173) and 919T>TG (chromosome 6: 43738364).Both mutations have a prevalence rate of 12.5%.These mutations have not been previously reported in external databases.The effects of these new mutations on the structure and function of the VEGF protein are yet unclear.Therefore, these findings provide an opportunity to explore novel therapies for GBM.
The involvement of mutations in the CTH gene has been proposed in several types of cancer [9,10], but their precise contribution to the formation of GBM is still not well understood.The study of the CTH gene in GBM patients identified a combination of silent mutations and mutations that result in changes to amino acids.Amino acid substitutions occurred in three out of the seven identified mutations: 28376T>TC, 28398A>AC, and 28399A>AT.The lack of prior documentation in external databases regarding these mutations underscores the possibility of finding new variants; the functional implications of these amino acid substitutions remain unknown.The remaining four mutations (28495A>A, 284400G>GT, 28577T>T, and 28611C>C) were silent mutations.Despite their generally lower probability of significantly affecting protein function, silent mutations can still influence splicing, mRNA stability, or protein expression levels [23].
The GENT2 database's analysis of gene expression showed a clear and distinct trend in GBM patients, particularly the elevated expression of TP53 and VEGFA genes.This finding seems paradoxical for TP53, a gene that typically inhibits tumor growth and is usually suppressed in cancer [24].However, cellular stress or DNA damage can upregulate TP53 despite the presence of inactivating mutations that render it nonfunctional [25,26].To understand the role of TP53 in the current scenario, additional studies into its mutational state and how it works within GBM patients are required.However, there is widespread recognition that GBM elevates VEGFA [27].This finding is consistent with previous investigations and emphasizes the continual importance of VEGFA signaling pathways as possible targets for treatment in GBM [28].CTH expression in GBM showed a modest but statistically significant increase.Although the exact function of CTH in GBM is still unclear, its possible significance in cell adhesion and migration justifies additional investigation, especially in connection with GBM invasion [29].These findings indicate an intricate interaction between variations in gene expression and the progression of GBM; therefore, further study is required to examine the practical effects of these alterations in expression and their potential as targets for medical treatment.
The study's limitations include a restricted GBM patient sample, which may limit the application of the findings to larger groups.Furthermore, the study failed to account for possible confounding factors such as comorbidities or medication usage, which might explain the observed variations in blood parameters and genetic abnormalities.

Conclusions
This study identified substantial changes in immune cell composition and inflammatory marker levels in GBM patients compared to the control group.The findings suggest a tumor microenvironment in GBM that promotes inflammation and suppresses immunological responses.Gene expression study found higher TP53 and VEGFA activity in GBM patients, possibly attributable to non-functional mutations.Further examination into the TP53 mutational status is required.The work underscores the relevance of VEGFA signaling pathways as therapeutic targets in GBM.CTH expression showed a modest increase in GBM, hinting at its potential role in GBM formation, necessitating further research.In conclusion, this study provides insight into the complex relationships between immune dysregulation, inflammation, and gene expression changes in GBM, opening the way for future strategies for therapy.

FIGURE 1 :
FIGURE 1: Comparison between the total number of WBCs, neutrophils, lymphocytes, and monocytes in healthy individuals and patients with GBM (A) No significant changes were observed in the total number of WBCs.(B) The percentage of neutrophils was significantly increased in patients with GBM compared to healthy individuals.(C) The percentage of lymphocytes was significantly decreased in patients with GBM compared to healthy individuals.(D) The percentage of monocytes was significantly increased in patients with GBM compared to healthy individuals.The comparison was performed using an unpaired t-test.*p<0.05;**p<0.01 vs. healthy individuals; GBM: glioblastoma; WBC: white blood cell

FIGURE 2 :
FIGURE 2: Liver function markers and CRP levels in healthy individuals and GBM patients No significant changes were observed in the AST (IU/L) (A), ALT (IU/L) (B), and ALP (IU/L) (C) in patients with GBM compared to healthy individuals.(D) The level of CRP (mg/L) was significantly increased in patients with GBM compared to healthy individuals.The comparison was performed using the Mann-Whitney test.**p<0.01 vs. healthy individuals; GBM: glioblastoma; ALT: alanine transaminase; AST: aspartate transaminase; ALP: alkaline phosphatase; CRP: C-reactive protein

FIGURE 3 :
FIGURE 3: TP53, VEGFA, and CTH gene expression in healthy individuals and patients with GBM retrieved from the GENT2 database.The log2 expression values of TP53 (A), VEGFA (B), and CTH (C) genes were significantly increased in patients with GBM compared to healthy individuals.The comparison was performed using an unpaired t-test.**p<0.01;****p<0.001 vs. healthy individuals; GBM: glioblastoma

TABLE 2 : Summary of mutations in the TP53, VEGFA, and CTH genes in GBM retrieved from the ICGC database.
GBM: glioblastoma; ICGC: International Cancer Genome Consortium