The Analysis of Polymorphism in DNMT3B Gene and Estimation of Their Effect on Tumor Phenotype and Prognosis on Breast Cancer Patients

tikslai:Mūsų tyrimo tikslas yra nustatyti genotipus ir alelius, susijusius su DMNT3B polimorfizmu. Be to, mūsų tyrimai apima bet kokio galimo ryšio tarp rs6058885 arba rs6058896 ir naviko augimo formos paiešką ir, dar geriau, ryšio tarp šių DNR variantų ir visos krūties vėžiu sergančių pacientų prognozės paiešką. Šis tyrimas rodo, kad pacientės ir gydytojo kova su krūties vėžiu vystosi su revoliucingesniais atradimais nei bet kada anksčiau Išvados ir rekomendacijos: DNMT3B variantų genotipiniai ir aleliniai dažniai - rs6058896 (CC: 87%, CT: 12%, TT: 1%; Alelis C: 93%, T: 7%) ir rs6058885 (CC: 87%, CT: 12%, TT: 1%; Alelis C: 93%, T: 7%)-neatskleidė reikšmingos koreliacijos su naviko patomorfologinėmis savybėmis. Be to, DNMT3B polimorfizmas nebuvo reikšmingai susijęs su krūties vėžio prognoze. Išsamūs tyrimai, apimantys didesnes kohortas, įvairias grupes ir platų genotipo nustatymą, yra pagrįsti siekiant išsiaiškinti galimą DNMT3B poveikį krūties vėžio rizikai ir rezultatams. Būsimi tyrimai turėtų apimti kitus genetinius ir epigenetinius parametrus, kad būtų išaiškintas jų sudėtingas ryšys su krūties vėžio patobiologija.

Background and objectives: At the time when science is moving fast giving those with breast cancer hope, it is still the reality that for those 100 women diagnosed, 17 still lose the battle because they have both fixed and adjustable risk factors. Some of them are molecular changes, for instance, the genes like BRCA1 and BRCA2 that are associated with genetic predisposition for cancer. Besides them, epigenetic changes such as DNA methylation that alter the normal functions of the genes that prevent the occurrence of cancer are also worth mentioning. Of these epigenetic mechanisms, DNA methylation stands out because of several enzymes including DNMT1, DNMT3A, DNMT3B. These enzymes are implicated in the onset of breast cancer and specifically DNMT3B is thought to be a particularly relevant molecular marker in this condition. Taking into consideration a large quantity of SNPs in the DNTM3B gene that are insufficiently investigated, our study examines the SNP variants rs6058885 and rs6058896. The purpose of our research is to identify genotypes and alleles associated with DMNT3B polymorphism. In addition, our research includes finding any possible relationship between rs6058885 or rs6058896 and the form of growth of a tumor and, even better, searching for the connection between these DNA variants and the entire prognosis for breast cancer patients. This study points to an evolution in the patient and clinician’s battle against breast cancer with more revolutionary discoveries than ever.

Material (patients) and research method used: Blood sample procurement for the breast cancer research took place at LUHS Institute of Oncology, between 2010 and 2017. Of the 171 patients enrolled in the study, strict standardized inclusion and exclusion criteria were used; patients needed to have valid diagnoses together with their medical records and those who had other cancers, other major comorbidities, and no sufficient documentation were excluded. Isolation of genomic DNA from the specimens was done using the "Gene JET Genomic DNA Purification kit" based on instructions from the kit manufacturer. Next, we conducted genotyping of DNMT3B polymorphisms rs6058896 and rs6058885 by RT-PCR using TaqMan SNP Genotyping Assays. The sample was standardized in concentrations of 10 μg/ml and 2 μg/ml, respectively, according to their volume. Water nuclease-free served as the negative control.

Findings/ results in sufficient details to support conclusions: In this study, we examined the polymorphisms rs6058896 and rs6058885 within the DNMT3B gene. Our findings indicate a consistent distribution pattern for both genetic variations; the CC genotype emerged as the predominant variant, succeeded by the CT and TT genotypes respectively. Notably, the genotype and allele frequencies mirrored one another, suggesting that these single nucleotide polymorphisms (SNPs) are in tight linkage disequilibrium; hence, their analysis was conducted concurrently. Conformity to Hardy-Weinberg equilibrium was confirmed for genotypes of DNMT3B polymorphisms rs6058896 and rs6058885. Subsequent statistical evaluation revealed no significant associations between these DNMT3B polymorphisms (both genotypic and allelic forms) and various pathomorphological features of tumors, such as age at diagnosis (P=0.368), tumor size (P=0.504), lymph node involvement (P=0.368), presence of metastasis (P=0.321), grade of differentiation (P=0.128), incidence of metastasis (P=0.321), progression of disease (P=0.595), and patient mortality (P=0.141). The analytical tools utilized for association assessment included Pearson’s Chi-square test or Fisher’s exact test, as appropriate. Furthermore, we conducted survival analyses on all investigated polymorphisms to estimate their prognostic significance in relation to disease outcomes. These survival analyses considered metastasis-free survival (hazard ratio [HR] – 1.677; 95% confidence limits [CL] – 0.437-1.197; P – 0.23), progression-free survival (HR – 0.1; 95% CL – 0.586-1.471; P – 0.752), and overall patient survival (HR – 0.903; 95% CL – 0.505-1.267; P – 0.752). These assessments were performed employing the Kaplan-Meier method with the Log Rank test (p > 0.05) and Cox regression analysis to determine statistical significance.

Conclusions and recommendations: Genotypic and allelic frequencies of DNMT3B variants—rs6058896 (CC: 87%, CT: 12%, TT: 1%; Allele C: 93%, T: 7%) and rs6058885 (CC: 87%, CT: 12%, TT: 1%; Allele C: 93%, T: 7%)—revealed no considerable correlation with tumor pathomorphological features. Additionally, DNMT3B polymorphisms were not significantly linked to breast cancer prognosis. Comprehensive studies, encompassing larger cohorts, diverse groups, and extensive genotyping, are warranted to elucidate DNMT3B's potential impact on breast cancer risk and outcome. Future investigations should integrate other genetic and epigenetic parameters to elucidate their complex relationship with breast cancer pathobiology.