Analysis of TP53 gene in breast cancer and comparison with the canine counterparts

2.1 Clinical evaluation and patient selection

Initially, a thorough search was conducted to identify families and individuals affected by breast cancer. The selection of breast cancer patients was based on the clinical information provided. These patients underwent careful examination by experienced oncologists to determine their tumor types, which were classified as showed in Table 1.

2.2 Source of sample and storage

Blood samples from ten patients diagnosed with breast cancer by a skilled phlebotomist. The blood was drawn into vacutainer blood collection tubes containing 0.5 M EDTA (ethylene diamine tetra acetic acid) as an anticoagulant. Blood samples were carefully stored at -20°C.

2.3 DNA extraction and genomic DNA estimation

DNA extraction was performed from preserved samples using a well-established method based on the protocol described by Sambrook and Russell, (2006). Following DNA extraction, gel electrophoresis was carried out to analyze polymerase chain reaction (PCR) products, adhering to the method previously outlined in (Hussussian et al., 1994). For DNA quantification, a nanodrop spectrophotometer (nanodrop 2000, thermo Scientific) was used, following the procedure described by Desjardins and Conklin et al. (Desjardins and Conklin, 2010). Initially, the nanoDrop instrument was prepared by placing 1 µL of nuclease-free water onto the pedestal to perform a blank measurement. This blank served as the baseline standard to calibrate the device and eliminate background absorbance. Once the blank was recorded, 1 µL of the extracted DNA sample, dissolved in nuclease-free water, was placed on the nanodrop arm. The sample was then analyzed by selecting the “measure” option on the connected computer, and the DNA concentration and purity were determined based on absorbance at 260 nm and 280 nm. This approach ensured accurate and reliable DNA quantification, critical for downstream molecular analyses.

2.4 Primer designing, synthesis and dilutions

Primer 3 software (https://bioinfo.ut.ee/primer3-0.4.0/) was employed to design primers that targeted the amplification of specific regions within the TP53 gene (Table 2). Specifically, the software was utilized to design primers for the amplification of Exon 5, which spans approximately 371 base pairs, and Exon 7, which encompasses around 299 base pairs of the TP53 gene sequence. This genetic sequence is readily accessible on the National Center for Biotechnology Information (NCBI) platform under the Accession number NC_000017.11. The TP53 gene is situated on chromosome number 17. This approach facilitated the precise targeting of these gene segments for further analysis and experimentation. Exon 5 and Exon 7 primers were synthesized by Gene Link under the brand e-oligos. The primers were obtained in lyophilized form, with concentrations measured in nanomoles. The initial concentration of each primer in stock was 100 picomoles, while the concentration of primers used in the actual experiments was 10 picomoles. The primers were appropriately diluted using a standard dilution method. Primers were checked for self-complementarity through oligocalc: Oligonucleotide properties calculator (http://biotools.nubic.northwestern.edu/OligoCalc.html). To check the specificity and non-specificity of primers, In silico PCR of UCSC (University of California Santa Cruz) genome browser (https://genome.ucsc.edu/) was used forward and reverse primers were put on In silico PCR and click on submit. This method gave amplified product of specific size.

2.5 PCR optimization

PCR optimization for Exon 5 and Exon 7 was systematically performed by using BIO-RAD T100^TM thermal cycler (Serial no. 621BR10685) (Made in Singapore). These optimization steps were carried out on control samples initially. To enhance the PCR conditions, adjustments were made to several factors including the concentration of magnesium ions (Mg²⁺), deoxyribonucleotide triphosphates (dNTPs), primers, and Taq DNA polymerase. These adjustments were tested across different temperature profiles. The experimental process involved the use of a primer set consisting of forward and reverse primers, Taq DNA polymerase, PCR buffer, dNTPs, MgCl₂, genomic DNA as the template, and nuclease-free water. Various conditions were explored to refine the amplification of the target Exons. The initial concentration of the control DNA sample was measured at 194.81 ng/µL, which served as the basis for the optimization of Exon 5 and Exon 7 amplification. Using the optimal concentration determined from the control DNA sample, the patient DNA samples were appropriately diluted. This ensured that the PCR amplification of Exon 5 and Exon 7 could be effectively tailored to specific characteristics of the patient samples.

2.6 Standard PCR of exon 5 and exon 7

The DNA samples underwent amplification using the standard PCR method at a temperature of 62°C. The amplification of Exon 5 and 7 was carried out using the BioRad PCR Thermocycler with both forward and reverse primers. The PCR reaction mixture consisted of a DNA template, forward and reverse primers, a 10X Taq Buffer containing Tris HCl-pH 8.8, (NH₄)₂SO₄, and Tween 20 (provided by Thermo Scientific®), MgCl₂ (also from Thermo Scientific®), dNTPs obtained from Thermo Scientific®, and molecular-grade water sourced from Getz Pharma®. The specific conditions for the Standard PCR procedure are mentioned in Fig. 1.

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Fig. 1.

Protocol for standard PCR of exon 5 and 7 primers.

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The PCR amplified product was subjected to a precipitation protocol for further processing. Initially, all the PCR amplicons were carefully transferred into individual 1.5 mL tubes provided by Biologix Group Limited®. To facilitate precipitation, approximately 100 µL of 80% ethanol sourced from AnalaR® was introduced into each tube, ensuring that the tube lids were closed. The tubes were subsequently shielded from light using aluminum foil from Diamond® as ethanol is known to be most effective under dark conditions. This arrangement was maintained for 30 min at a temperature of 4°C. Following the 30 min incubation, the tubes were unveiled by removing the aluminum foil. To achieve the precipitation step, centrifugation was carried out at 4500 rpm for 20 min of 4°C. The supernatant resulting from centrifugation was carefully discarded, leaving behind a DNA pellet. This pellet was allowed to air dry until the noticeable scent of ethanol from AnalaR® dissipated. To proceed, 15 µL of nuclease-free water supplied by Getz Pharma® was introduced to the dried DNA pellet. With the DNA now resuspended, the subsequent step involved conducting gel electrophoresis. A 1.2% agarose gel was meticulously prepared, and an electrical potential of 110 volts was applied for a duration of 25 min to facilitate the migration of DNA fragments. After the gel electrophoresis process was completed, the gel was transferred to a Biorad Gel Documentation system to captured images for further analysis.

2.7 Sequencing of amplified product

Following the precipitation of the PCR product, the sequencing procedure was initiated, employing the Sanger chain termination, also known as dideoxy termination, method (Sanger, Nicklen and Coulson, 1977). This method relies on several essential components, including DNA polymerase, the target DNA template, fluorescently labelled nucleotides dNTPs, a primer, and modified nucleotides ddNTPs that lead to termination of chain elongation. To accomplish the sequencing, a series of four distinct reactions were set up, each utilizing the same DNA template. Within the sequencing reaction mixture, crucial ingredients encompass the DNA template, DNA polymerase, primers, and the necessary deoxynucleotide triphosphates (dTTP, dATP, dCTP, dGTP). Additionally, dideoxy nucleotide triphosphates (ddTTP, ddATP, ddCTP, ddGTP) were included in each reaction. It is notable that these dideoxy nucleotides (ddNTPs) were characterized by their lack of a 3’OH group, a pivotal element required for the formation of phosphodiester linkages, as emphasized by Sanger et al. (1977) (Sanger, Nicklen and Coulson, 1977). This absence of the 3’OH group in ddNTPs results in the termination of chain elongation due to the inability to form phosphodiester bonds, effectively halting further extension of the DNA strand. For the actual sequencing process, the samples were subjected to analysis using the ABI 3130 XL genetic sequencer/analyzer (Applied Biosystems, Lahore city, Advance Bioscience international Pakistan). The reagents employed in the sequencing reaction are enumerated in Table 3:

Following PCR product sequencing, the samples were precipitated using 75% ethanol, with 40 µl ethanol added to each reaction, resulting in a final concentration of 60%. The mixture was vortexed and left at room temperature for 20 min. Centrifugation was then performed at 14,000 rpm for 20 min at 4°C. After centrifugation, the supernatant was discarded, and the pellet was washed with 100 µl of 70% ethanol. Following washing, the pellet was air-dried at room temperature. Subsequently, 15 µL of deionized formamide was added to each pellet. Denaturation was carried out at 95°C for 5 min, followed by chilling before loading onto the ABI 3130 genetic analyzer. Analysis was conducted using ABI PRISM sequencing analysis software version 3.7. Conditions and protocol are shown in Table 4 below.

2.8 Data analysis of amplicon

Bioinformatics tools were employed for the comprehensive analysis of the amplified sequences. For manual sequence editing and alignment, bioedit software (Version 7.2.5) was utilized. To analyze nucleotide sequence homology, the NCBI nucleotide (BLAST) basic local alignment search tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was employed (Altschul et al., 1990), which allowed for the comparison of the amplified sequences against nucleotide sequences in the NCBI database. Additionally, sequences were aligned against the reference sequence using BLAST software for more precise comparison and analysis (Wheeler et al., 2008). Chromas software was used to interpret sequencing results, including the examination of chromatogram peaks and reading of nucleotide sequences. For the detection and classification of mutations or variations in the TP53 gene, the Mutation Tester online software (https://www.genecascade.org/MutationTaster2021/#transcript) was applied to identify novel or previously reported mutations.

To evaluate differences in codon usage and amino acid sequences between normal and mutated proteins, the EXPASY (https://web.expasy.org/translate/). was employed. For analyzing the primary structure of mutated proteins, the Protter online tool (https://wlab.ethz.ch/protter/start/) was utilized to visualize the protein sequence and structural domains. Finally, to construct the three-dimensional structure mutated proteins, trRosetta software (https://yanglab.qd.sdu.edu.cn/trRosetta/) was used. This tool enabled the design of high-confidence 3D models, providing a detailed structural comparison of normal and mutated TP53 proteins for further functional analysis.

2.9 Statistical analysis

Mutation data obtained from the sequencing analyses were statistically evaluated using the SNPStats online software (https://www.snpstats.net/). This software provides comprehensive statistical approaches specifically tailored to analyze SNPs, allowing for the assessment of allele frequencies, genotype distribution, association studies, and potential correlations with clinical or pathological characteristics. Previous studies, including those by Elsaid et al., (2017) and Youssef et al., (2021), have demonstrated the reliability and robustness of SNPStats software in analyzing genetic variations, including its ability to generate odds ratios with corresponding 95% confidence intervals, further supporting its suitability for this study statistical analyses.

2.10 Homology analysis of homo sapiens (Human) and cannis lupus (Dog)

The nucleotide sequences of human and dog Exon 5 and Exon 7 regions were retrieved from the NCBI database. Sequence conservation between species was assessed using the BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi), applying default parameters for nucleotide-nucleotide comparisons. Alignments were examined for sequence identity, presence of gaps, and strand orientation. Conserved and mismatched positions were noted to identify potential SNPs or evolutionary differences.

3.1 DNA extraction and genomic DNA estimation

In this study, genomic DNA was extracted from blood samples of ten breast cancer patients using the PCI (phenol/chloroform/isoamyl alcohol) method. The extracted genomic DNA was assessed through both qualitative and quantitative methods.

For qualitative estimation, gel electrophoresis was employed to evaluate the integrity of DNA, as illustrated in Fig. 2, which shows the outcomes of samples obtained from breast cancer patients. The Thermofisher 1kb Plus DNA Ladder (Lot No. #10787026) was utilized.

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Fig. 2.

Gel electrophoresis of breast cancer patient samples.

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For quantitative estimation, a nanodrop Spectrophotometer 2000 was utilized to determine the concentration of genomic DNA. The optical density (OD) values were measured at a wavelength ratio of 260/280, with each sample exhibiting an OD value of approximately 1.8, indicative of pure genomic DNA. The detailed OD values for all the samples are provided in Table 5. These results confirm the successful extraction and quantification of genomic DNA, offering valuable insights into the DNA content in the tested samples.

3.2 Standard PCR of exon 5 and 7

A standard PCR protocol was employed to amplify Exon 5 and Exon 7. A total of 35 cycles were carried out, utilizing an annealing temperature of 62°C to achieve a specific product as depicted in Fig. 3.

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Fig. 3.

Standard PCR gel electrophoresis of (a, b) Exon 5 and 7.

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4.1 Chromatogram analysis of TP53 gene

The results of the chromatogram analysis revealing several genetic variations within the TP53 gene. As shown in Fig.4 panel (a) shows a substitution where adenine (A) is replaced by guanine (G) in Intron 6, a genetic variation that may influence the splicing mechanism or the regulation of TP53 expression. Panel (b) highlights the deletion of cytosine (C) in Exon 4, which could potentially alter the coding sequence of the TP53 protein, leading to changes in its structure and function. Panel (c) depicts a cytosine (C) deletion in Intron 7, a variation in the non-coding region that might affect intron-exon splicing or gene regulatory elements. Finally, panel (d) reveals a deletion of adenine (A) in Intron 8, which could disrupt the normal splicing process or influence the gene’s transcriptional regulation. These genetic alterations, identified through chromatogram analysis, could have significant implications for the TP53 gene’s function, potentially impairing its tumor-suppressing role and contributing to cancer development.

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Fig. 4.

The chromatogram analysis results revealed the following genetic variation: (a) Adenine replaced by Guanine (A>G) in intron 6, (b) Deletion of C in Exon 4, (c) Deletion of C in intron 7 and (d) Deletion of A in intron 8. (Arrow shows deletion and replacement)

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4.2 Blast of reference and mutated protein

The reference protein of TP53 NP-000537.3 has 393 aa, and mutated protein has 245aa. Protein blast of both proteins showed homology of 177 amino acids. As shown in Fig. 5, the sequence of amino acid after 178 aa got change and truncated protein was formed due to early stop codon at position 246 aa.

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Fig. 5.

Blast analysis of normal and mutated TP53 protein.

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The results demonstrate the consequences of a frameshift mutation on the protein-coding sequence, which was analyzed using the Expasy Translate tool. This mutation involves the deletion of a single nucleotide, which disrupts the original reading frame of the gene. As a result, the amino acid sequence remains unchanged up to position 177aa, after which the sequence shifts, producing a completely altered and nonfunctional amino acid chain.

As you can see in Fig. 6 frameshift mutation introduces an early stop codon at position 246aa. The presence of this premature stop codon results in the production of a truncated protein that is significantly shorter than the original, leading to the loss of critical functional domains required for normal protein activity. The Fig. 6 clearly illustrates this disruption, showing the abrupt change in the amino acid sequence following position 177aa and highlighting the early termination of translation. The frameshift mutation not only alters the sequence of amino acids but also affects the overall structure and stability of the protein. Such structural changes can render the protein nonfunctional or even harmful to the cell, potentially disrupting normal biological processes. This finding underscores the significance of maintaining the correct reading frame for the synthesis of a functional protein and highlights the detrimental impact of frameshift mutations on gene expression and protein function.

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Fig. 6.

Frameshift mutation leading to altered amino acid sequence and early stop codon at position 246.

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The 3D structure of the mutated TP53 gene is generated using trRosetta, as shown in Fig. 7(a). The primary structure of the mutated protein was modeled using the Protter tool, which provides a visual representation of transmembrane proteins. The illustration highlights the impact of the frameshift mutation on the protein’s structure. As shown in the Fig. 7(b), the frameshift mutation causes a significant alteration in the amino acid sequence starting from position 177aa, disrupting the normal structure. This mutation results in the formation of a truncated protein, terminating prematurely at position 246aa due to the introduction of a premature stop codon. The truncated protein is shown as lacking key functional domains, including critical transmembrane regions, which are essential for proper folding, stability, and function. The annotations in the Fig. 7 mark key features such as the N-glycosylation motifs (green circles), signal peptides (red crosses), and transmembrane regions (indicated by loops). The altered amino acid sequence and the premature termination are evident, demonstrating that the mutation not only disrupts the protein’s structural integrity but also renders it nonfunctional. This result underscores the critical importance of maintaining the correct reading frame to preserve protein functionality. This visual model helps to better understand the detrimental impact of this mutation at the molecular level, particularly in how it disrupts the protein’s ability to span membranes and perform its normal biological roles.

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Fig. 7.

(a) 3D structure of the mutated TP53 protein, highlighting the structural alterations resulting from the variation. (b) Primary structure of the mutated TP53 showing the sequence of amino acids and the specific mutation sites.

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The secondary structure of the mutated TP53 protein was predicted using PSIPRED, a widely used tool for protein structure prediction (Fig. 8). PSIPRED employs a highly accurate, two-stage method that integrates information from multiple sequence alignments and predicts the locations of alpha helices, beta strands, and coils in the protein. This approach enables PSIPRED to provide reliable predictions even for sequences with limited homology to known structures. By applying PSIPRED to the mutated TP53 protein, we can visualize the structural changes resulting from the mutation, which may alter its functional domains and stability. This secondary structure prediction offers valuable insights into the potential effects of mutations on the protein’s folding and function, aiding in the understanding of its role in cancer development and progression.

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Fig. 8.

(a) Prediction of the secondary structure of the mutated TP53 protein, illustrating the arrangement of alpha helices, beta sheets, and loops, (b) Highlighted domains within the mutated TP53 protein, emphasizing functional regions affected by the mutation.

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4.3 Statistical analysis

• 1. Deletion of C present only in 1 count. The allelic frequency for 1 count is 0.05 for cancer samples. The genotypic frequency for the C/- genotype is 0.1 in cancer samples. About 10% of cancer samples have the C/- genotype. The exact test for Hardy-Weinberg equilibrium for genotype C/- in cancer samples showed a non-significant result; p-value = 1 with 95% confidence interval. This change is not significantly associated with cancer; p-value = 0.23.

• 2. The two alleles for second nucleotide variant are A and G. Allele G is homozygous, present in 4 counts. The allelic frequency for 4 counts is 0.2 for cancer samples. The genotypic frequency for the G/G genotype is 0.8 in cancer samples. About 40% of cancer samples have the G/G genotype. The exact test for Hardy-Weinberg equilibrium for genotype G/G in cancer samples showed a non-significant result; p-value = 1 with 95% confidence interval. This change is significantly associated with cancer; p-value = 0.01.

• 3. Deletion of C present only in present in 6 counts. The allelic frequency for 6 counts is 0.3 for cancer samples. The genotypic frequency for the C/- genotype is 0.6 in cancer samples. About 60% of cancer samples have the C/- genotype. The exact test for Hardy-Weinberg equilibrium for genotype C/- in cancer samples showed a non-significant result; p-value = 0.48 with 95% confidence interval. This change is significantly associated with cancer; p-value = 0.0009.

• 4. Deletion of A present only present in 1 count. The allelic frequency for 1 count is 0.05 for cancer samples. The genotypic frequency for the A/- genotype is 0.1 in cancer samples. About 10% of cancer samples have the A/C genotype. The exact test for Hardy-Weinberg equilibrium for genotype A/- in cancer samples showed a non-significant result; p-value = 1 with 95% confidence interval. This change is not significantly associated with cancer; p-value = 0.23.

4.4 Homology analysis of homo sapiens (Human) and cannis lupus (Dog)

The BLAST analysis results demonstrate significant sequence conservation between human and dog Exon 5 and Exon 7 regions, as shown in Fig. 9. Exon 5 shows a high alignment score of 146 bits with an E-value of 2e-40, indicating a highly significant match. The sequence identity is 88% (100 out of 113 nucleotides), with no gaps observed, highlighting a continuous and well-aligned region in the same orientation (Plus/Plus strand). In comparison, Exon 7 also exhibits strong conservation, with a score of 135 bits and an E-value of 5e-37. The identity is slightly lower at 83% (106 out of 127 nucleotides), but like Exon 5, it has no gaps and aligns in the same orientation. The yellow highlights in both alignments mark mismatched positions, which likely represent SNPs or evolutionary differences. Overall, Exon 5 appears to be more conserved than Exon 7, with both exons showing no major structural differences, emphasizing their evolutionary significance across species.

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Fig. 9.

(a, b) Comparative homology analysis of Homo sapiens and Canis lupus TP53 exon 5 and exon 7.

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