|Year : 2017 | Volume
| Issue : 2 | Page : 426-429
Microsatellite Instability in Chronic Myeloid Leukemia using D17S261 and D3S643 markers: A Pilot Study in Gujarat Population
TN Patel1, M Chakraborty2, P Bhattacharya1
1 Departments of Integrative Biology, Vellore Institute of Technology, Vellore, Tamil Nadu, India
2 Departments of Biomedical Genetics, Vellore Institute of Technology, Vellore, Tamil Nadu, India
|Date of Web Publication||21-Feb-2018|
Dr. T N Patel
Departments of Integrative Biology, Vellore Institute of Technology, Vellore, Tamil Nadu
Source of Support: None, Conflict of Interest: None
CONTEXT: Tumor progresses through a series of genetic alterations that involve proto-oncogenes and tumor suppressor genes – the gatekeeper, caretakers, and landscaper genes. Microsatellites are short tandem repeat sequences, present over the span of human genome and are known to be variable at multiple loci due to errors in DNA Mismatch Repair machinery. AIM: The present study was aimed to evaluate the association between Microsatellite Instability (MSI) and evolution of Chronic Myeloid Leukemia (CML) – genetically a rare event but profound in this pilot study. SETTINGS AND DESIGNS: We explore the possibility of MSI in primary CML patients confirmed by t(9;22) using capillary electrophoresis. Fifteen CML patients and healthy individual samples, respectively, were used to study the markers D17S261 and D3S643. MATERIALS AND METHODS: The DNA was amplified using tagged and nontagged primers and further subjected to bioanalysis and fragment analysis. RESULTS: While the results from bioanalyzer fluctuated, fragment analysis indicated the presence of microsatellite variability in 80% of the patients' samples as compared to no MSI in normal individuals for both the markers. CONCLUSION: These findings suggest that MSI is a genetic event that may have a role in CML progression or evolution. Further studies are warranted to understand the plausible underlying causes.
Keywords: Bioanalysis, Chronic Myeloid Leukemia, D17S261, D3S643, fragment analysis, Microsatellite Instability
|How to cite this article:|
Patel T N, Chakraborty M, Bhattacharya P. Microsatellite Instability in Chronic Myeloid Leukemia using D17S261 and D3S643 markers: A Pilot Study in Gujarat Population. Indian J Cancer 2017;54:426-9
|How to cite this URL:|
Patel T N, Chakraborty M, Bhattacharya P. Microsatellite Instability in Chronic Myeloid Leukemia using D17S261 and D3S643 markers: A Pilot Study in Gujarat Population. Indian J Cancer [serial online] 2017 [cited 2019 Nov 17];54:426-9. Available from: http://www.indianjcancer.com/text.asp?2017/54/2/426/225804
| » Introduction|| |
Chronic Myeloid Leukemia (CML) is a clonal myeloproliferative disorder of pluripotent stem cells involving erythroid, megakaryocyte, myeloid, B-lymphoid, and T-lymphoid elements. CML almost always evolves from a chronic disease and progresses toward a more destructive leukemia followed by accelerated phase and blast crisis phase., The characteristics of these stages are increased cellular proliferation and maturation arrest. The disease is invariably associated with the presence of Philadelphia chromosome (Ph) – t(9;22) (q34;q11), along with the molecular event of breakpoint cluster region-Abelson (BCR-ABL) fusion protein products. The ABL proto-oncogene is localized on 9q34, and BCR proto-oncogene is localized on 22q11. Molecularly, the translocation results in BCR-ABL fusion protein encoding 185–210 kD hybrid products with an increased tyrosine kinase activity. BCR-ABL tyrosine kinase activation is crucial for the pathogenesis of chronic phase of CML, but its conversion to blast crisis phase is unclear. In blast crisis, acquisition of newer cytogenetic anomalies such as + 8, +19, +21, i(17q), t(3;21)(q26; q22), t(15;17)(q24;q21), t(9;22;16)(q34;q11;q24), t(4;9;22)(q25;q34;q11), and t(9;10;22)(q34;p11.2;q11.2) in addition with Ph chromosome has been reported.,,
Genomic instability deals with the understanding of genome-wide changes that lead to functionally impaired genomic regions and complex disease conditions. Chromosomal instability is microscopically visible changes in the chromosomes whereas microsatellite instability (MSI) is usually accompanied by variations in the nucleotide repeats as a result of impaired DNA repair systems., Both these conditions lead to development and progression of cancers. Microsatellites are highly polymorphic, short repeat nucleotide sequences spread throughout the genome. The repetitive di- and tri-nucleotides are sited about once in every 40 kb of DNA between or within the genes. Simple sequence repeats (SSRs)/microsatellites can be (A)n, (CA)n, and (GATA)n, where n is between 5 and 30. In normal cells, the numbers of repeat unit are maintained with high accuracy within a genome while in cancers, these numbers of repeat unit become hypermutable and variable. MSI represents variations may be a consequence of replicative errors that are a result of defective Mismatch Repair (MMR) genes such as human MutS protein homolog 2 (MSH2), human MutL homolog 1 (MLH1), MSH6, and PMS1 homolog 1 (PMS2) [Figure 1]. Although many studies reported the presence of microsatellite instabilities in gastrointestinal cancer, colorectal cancer, breast cancer, endometrial cancer, head and neck squamous cell carcinoma, and sarcomas;,,, nonetheless, it is considered a rare event in hematologic cancers. In this study, we establish for the first time, the presence of MSI in primary cases of CML patients in India and in succeeding studies hope to prove this to be an underlying phenomenon in various hematologic cancers.
Of the multiple markers that are known in the human genome, in this study, MSI was investigated using D17S261 (Mfd41) and D3S643 (CI3-9) markers, and analysis of MSI was performed by comparing electropherograms for both normal and cancerous samples. The results indicated the presence of MSI for the markers examined.
| » Materials and Methods|| |
Fifteen CML – Ph + (cytogenetically confirmed) and control DNA samples were provided by Genexplore Diagnostics and Research Centre Pvt. Ltd., in MoU with VIT University as per the ethical guidelines. The diagnosis of CML was based on standard clinical criteria and cytogenetics report of t(9;22) positive in all the samples. The quality and quantity of DNA was checked in NanoDrop 2000 UV-Vis spectrophotometer and 0.8% agarose gel electrophoresis, respectively.
A set of primer pairs for D17S261and D3S643 with chromosomal loci for these markers on 17p and 3p, respectively, were synthesized from Shrimpex Biotech Services Pvt. Ltd., to conduct the study. The sequences of forward and reverse primers are given in [Table 1]. The reverse primers of both the markers were tagged with fluorescent dye FAM (D17S261) and HEX (D3S643), respectively, to carry out single reaction Capillary Electrophoresis (CE).
Standard Polymerase Chain Reaction (PCR) was performed using the set of predesigned work primers. Both control and patient DNAs were initially denatured at 96°C. For D17S261 and D3S643 primers, the annealing temperature was set as 50°C and 60°C (FAM-tagged D17S261 and HEX-tagged D3S643), respectively. The PCR amplified products were then electrophoresed on 2% agarose gel at voltage 50 V to confirm the presence of PCR products.
Agilent 2100 Bioanalyzer was used with high sensitivity DNA kit for performing bioanalysis. In gel loading wells (4 wells), 9 μl of gel-dye mix was added and subjected to pressure for 1 min to form a gel base. A 5 μl of high sensitivity DNA marker was pipetted into each of the ladder well (1 well) and sample wells (11 sample wells). A 1 μl of PCR product and 1 μl of ladder were added into the corresponding sample and ladder wells. All the experiments were carried out in duplicate. The chip was placed horizontally in the adapter of the vortex mixer for 60 s at 2400 rpm. Nuclease-free water was loaded in additional chip to wash electrodes manually inside the Agilent 2100 Bioanalyzer. The sample loaded chip was inserted.
Reaction mixture for single sample was 0.5 μl of PCR product, 0.5 μl of size standard (Liz 500-orange colored dye), and 9 μl of HI-DI formamide (denaturing agent). A Master Mix of the size standard and formamide was prepared. The samples and Master Mix were added to each well. The reaction mixture was denatured for 5 min at 95°C and snap freeze on ice for 2–3 min. Mixed samples were loaded in the CE Genetic Analyzer. The intensity of fluorescence was recorded as a function of wavelength and time on a charge coupled device camera. The data were analyzed with GeneMapper software [Manufacturer: Thermo Fisher Scientific].
| » Results|| |
The DNA samples for fifteen control and patients were subjected to PCR, and the products for tagged and nontagged primers were run on 2% agarose gel. The gel electrophoresis confirmed the presence of PCR products of 159 bp and 113 bp, respectively, for D17S261 and D3S643 markers. Typically, in the D3S643, there was a product of lower molecular weight that was co-amplified in the PCR and was also seen across as a constant peak at 83 bp in fragment analysis.
The lower marker (15 bp) and the upper marker (1500 bp) were the internal standards used to align the ladder data with the data obtained from the samples. Bioanalysis was performed to check the variation in the CA repeats in patient and control samples. The patient samples and control showed peaks in the standard size range of 153–172 bp for D17S261 (Mfd41) microsatellite marker [Figure 2]. For D3S643 (CI3-9) marker, both patients samples and control showed peaks in 120–130 bp region [Figure 3]. As the peaks of samples as well as control overlapped, peak image of one patient sample along with ladder is being provided to understand the analysis report. However, this is a quantitative assay; but for a confirmatory result, we looked in detail with fragment analysis.
|Figure 2: Bioanalysis result for patient sample along with ladder for D17S261 marker|
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|Figure 3: Bioanalysis result for patient sample along with ladder for D3S643 marker|
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Hypervariability was present in all the patients studied for (CA) repeats in the 159–161 bp region for D17S261 (Mfd41) marker [Figure 4]. For D3S643 (CI3-9) marker, about 40% (6) of patients did not show any variation and were similar to control sample. However, nine patients showed variability near 94 bp, 101 bp, and 100 bp indicating instability. A common peak of 85 bp was constant in all the 15 patients with D3S643 primers. This region was nonvulnerable constant repeat that was otherwise not reported in earlier studies with the same set of primers [Figure 5].
|Figure 4: Fragment analysis result of control and patient sample for D17S261 (Mfd-41) marker|
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|Figure 5: Fragment analysis result of control and patient sample for D3S643 (CI3-9) marker|
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| » Discussion|| |
The frequency of SSR is on an average one per 100,000 bp throughout the genome., In the present study, the patients showed distinct mutability in the number of CA repeats at the loci that were explored for the variation. The marker D17S261 was positive for MSI in all the patients screened; however, only 9 out of 15 patients showed microsatellite variation for D3S643 marker. The overall result does direct toward hypermutability of microsatellite which may impact the functioning of the proteins encoded by genes CMT1A duplicated region transcript 4 (CDRT4) (D17S261) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-4 (PFKFB4) (D3S643).
The D17S261 marker is specific to a protein-coding gene CDRT4, located at 17p12. In several myeloid and lymphoid cell lines, the expression of CDRT4 is seen either to be upregulated or downregulated though, in the cancers of the stomach and liver, this gene expression is extremely high. The CA repeats are within the protein-coding region of the gene, and hence, any change in the repeat number from normal affects the protein function. It can be theorized that due to repeat variability CDRT4 gene is acquiring insertion frameshift in DNA hence altering the protein sequence. The CDRT4 is further linked to Charcot-Marie-Tooth disease More Details type 1A (CMT1A) protein involved in inherited peripheral neuropathies, CMT1A and hereditary neuropathy with liability to pressure palsy. Although the actual function of CDRT4 is not known in cancer, it is prominently found to be upregulated in various cancers. It is already proved that MSI is instigated by mutational inactivation of DNA repair genes and a number of researches support the fact that SSR-containing unigene can be recurrently altered by MSI in tumor cells.
The D3S643 marker is specific for a protein-coding gene PFKFB4 which is overexpressed in cancer cells induced by hypoxia.,, The bifunctional enzyme PFKFB4 has distinct kinase: phosphatase activities. The presence of SSR within the coding regions of the protein indicates impaired protein function. Lin et al. show that loss of p53 and MMR genes cause an increased frequency of frameshift mutations. We understand that MSI caused due to MMR deficiency increase the PFKFB4 expression with increase reactive oxidative species thus activating angiogenesis and promoting tumor progression.
| » Conclusion|| |
In summary, D17S261 (Mfd41) and D3S643 (CI3-9) markers were chosen to study the incidence of MSI. These markers showed variation at their respective loci in 80% of the patients under the study. The study reveals both the markers may be consequence of altered DNA repair systems which in turn affects the CDRT4 and PFKFB4 gene functions. The MSI results using fragment analysis were more accurate and highly reproducible. MSI analysis could be cost-effective screening for t(9;22) positive patients who manifest crisis stage without secondary cytogenetic change thus indicative of mutations in DNA repair genes and genomic instability. The line of treatment for such cases could be decided by the diagnostic results of specific microsatellite markers. Since the sample size is very small, further studies are warranted with larger samples covering various hematologic malignancies and multiple SSR markers.
We are grateful to thank Dr. Rank and Dr. Koringa along with Mrs. Pooja and Ms. Shefali at College of Veterinary Sciences, AAU, Anand – Gujarat, for allowing us to conduct fragment analysis. We are also thankful to Dr. Alpesh and Dr. Shiva Sankar of Genexplore Diagnostic Laboratory, Ahmedabad for helping us with the sample collection and conducting bioanalysis studies.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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