GENETIC PREDICTORS OF TOXIC EFFECTS OF METHOTREXATE IN CANCER PATIENTS.

Today, methotrexate (MTX) is used in combination with other medicines to treat a wide range of malignancies. Despite its proven high efficacy, MTX often causes serious side effects, which may result in the need to reduce the dose of MTX or discontinue the drug altogether. This, in turn, can provoke the development of MTX resistance and cancer progression. Predicting the risk of MTX-induced toxicity is currently difficult due to the variability of pharmacokinetics and pharmacodynamics in different patients, so the scientific literature is intensively searching for potential biomarkers. Based on the data available in the current literature, we analyzed the relationship between variants in the genes encoding the key components of MTX intracellular metabolism and the MTX-induced side effects and drug response. According to the results of our work, the most studied variants are those of the SLC19A1 gene, which encodes the reduced folate carrier protein 1, and the MTHFR gene, which encodes the enzyme methylenetetrahydrofolate reductase. Studies of the effect of methylation of the promoter regions of genes on the therapeutic effect of MTX are also very promising. In conclusion, the study of molecular genetic markers of MTX toxicity is extremely relevant and necessary because it can help to avoid the effect of multidrug resistance and improve the quality of life and survival of patients.

CLINICAL SIGNIFICANCE OF BRCA1 GENE SEQUENCING AND ITS PROMOTER METHYLATION TESTING IN THE SEARCH STRATEGY FOR THERAPEUTIC TARGETS IN BREAST CANCER TREATMENT.

BACKGROUND: Currently, there is a great interest in the genetic testing of BRCA1 and BRCA2 due to the fact that for patients with breast cancer (BC) with pathogenic variants of these genes, the use of the PARP inhibitors could be also provided in addition to implemented treatment protocols. The aim of this study was to characterize the molecular genetic structure of the BRCA1 gene in BC patients without progenitor germline mutations taking into account the methylation state of the promoter region. MATERIALS AND METHODS: The study involved 210 patients with newly diagnosed BC. The most common germline pathogenic variants of the BRCA1 (185delAG, 5382insC, 4153delA, T300G) and BRCA2 (6174delT) genes were identified in the peripheral blood. A subgroup of 14 patients without progenitor pathological variants of the BRCA1 and BRCA2 genes and with a family history of cancer was randomly selected. For them, BRCA1 gene sequencing by Sanger and hypermethylation of the BRCA1 gene promoter region were analyzed. RESULTS: The following frequencies of BRCA1 mutations were determined in the general group: 5382insC - 8.6%, 4153delA - 0.5%, T300G - 0.5%. The analysis of the BRCA1 gene by Sanger sequencing revealed 11 BRCA1 gene variants in 10 out of 14 BC patients. All of them, according to the currently available data, were defined as "benign" and not clinically relevant. The frequency of the detection of hypermethylation of the BRCA1 gene promoter region in the randomly selected group of patients was 14.3%. CONCLUSIONS: In BC patients, not only common mutations but also the methylation status of the BRCA1 gene promoter region in the peripheral blood should be determined. The whole-genome sequencing of the BRCA1 gene may be the last step in determining the genetic characteristics of BC patients carried out to optimize the treatment and improve survival thanks to the higher prevalence of the progenitor mutations and hypermethylation of the BRCA1 gene promoter.

Associations of variants of folate cycle genes with features of the clinical course of severe intraventricular hemorrhages in premature infants.

BACKGROUND: Summary data indicate that it has increased attention to the study of the role of the folate cycle and the genes encoding its key components in the complicated course of the neonatal period in premature infants. Therefore, the aim of our study was to investigate the relationship of folate cycle gene variants with the features of the neonatal course in premature infants with severe intraventricular hemorrhages (IVH). METHODS: The study included 24 preterm infants with with IVHs of 3d and 4th degree that received standard clinical, laboratory and instrumental examination. RESULTS: Apgar scores at 1 and 5 minutes were significantly lower in patients with AA genotype according to variant A1298C of the MTHFR gene. The concentration of total protein on 6th day after birth was negatively correlated with the A66G variant of the MTRR gene. The mean concentration of ionized calcium in the first day after birth was higher in the subgroup of patients with the AA genotype (according to variant A1298C of the MTHFR gene). In the subgroup of patients requiring mechanical ventilation, the frequency of AA genotype according to variant A2756G of the MTR gene was significantly increased. The presence of respiratory disorders and oxygen dependence was negatively correlated with variant A1298C MTHFR. The day of surfactant administration was positively correlated with variant A1298C of the MTHFR gene. CONCLUSION: The results of this study indicate that gene variants MTHFR (C677T, A1298C), MTRR (A66G), MTR (A2756G), RFC1 (G80A) may affect the neonatal course in premature infants with severe IVH.

CYP2C19*2 gene variant (G681A, rs4244285) as a prognostic marker for the clinical course of multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is the most common type of paraproteinemic hemoblastosis, which is characterized by an aggressive course, high mortality and a large number of complications. The G681A variant (*2, rs4244285) of the CYP2C19 gene leads to the formation of an inactive enzyme and, as a consequence, may affect the development and course of MM. The aim of this research was to analyze the effect of the G681A variant of the CYP2C19 gene on the risk of the development of MM and its course. MATERIALS AND METHODS: The study enrolled 158 patients with MM, who underwent standard clinical and laboratory studies: cytological, general clinical, biochemical, as well as molecular cytogenetic and molecular genetic. Cytogenetic analysis of chromosome abnormalities was performed using interphase fluorescence in situ hybridization. Genotyping by the G681A variant of the CYP2C19 gene was performed by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: No association was found between the G681A variant of the CYP2C19 gene and the risk of developing MM. The association between the presence of the G allele and GG genotypes with significant changes in clinical and biochemical parameters (plasma cell count, α2-globulin, calcium content) in MM patients has been established. In the presence of the G allele of the CYP2C19 gene, the development of chromosomal rearrangements del(13q14.2) or del(13q34) with significantly increased levels of albumin occurs more frequently. CONCLUSIONS: The G681A variant of the CYP2C19 gene does not affect the risk of developing MM, but it is associated with significant changes in the clinical and biochemical parameters that determine the severity of the disease and its prognosis. Further research is important to develop new target strategies and maintenance therapy for carriers of different variants of the CYP2C19 gene (G681A).

VARIANTS OF IL1 (C3954T, RS1143634), PON1 (C108T, RS705379) GENES AS PROGNOSTIC MARKERS OF OSTEOMYELITIS RISK AND ITS COMPLICATIONS.

The aim of the work was to study the effect of IL1Β (C3954T, rs1143634), PON1 (C108T, rs705379) gene variants on the risk of bacterial osteomyelitis development and its complicated course. The study involved 56 patients with osteomyelitis - 20 with not complicated (non-recurrent) course after treatment and 36 with complicated (recurrent) course. The data of population frequencies for the European population, obtained from the open database of "1000 Genomes project", were used as a control group. There was significantly increased distribution frequency of genotype 3954TT of IL1Β in patients with uncomplicated course compared to the control group (χ2=6.05, p=0.014, OR=4.99 (1.55-16.07)). And was found increased of minor genotype 108TT of PON1 frequency in patients with osteomyelitis compared to control group (χ2=4.38, p=0.036, OR=1.85(1.03-3.33)). There were found gender differences in the clinical effects of IL1Β gene variant: in men, the prevalence of genotype 3954CC was significantly to be increased in the patient with complicated osteomyelitis; genotype 3954CT was associated with a reduced risk of osteomyelitis and its complications developing, while in women was found the association of genotype 3954TT with an uncomplicated course of the disease. In conclusion, this study suggests that the variants of IL1В and PON1 genes associated with the risk of developing bacterial osteomyelitis and its complicated course and can be used as a prognostic marker for developing personalized prevention strategies.

ESR1 gene variants affect FSHR-depended risk of fibrocystic mastopathy in infertile women.

BACKGROUND: The infertile women have an increased risk of developing benign and malignant tumors, in particular, breast cancer. Most studies have examined the role of gene variants in the risk of developing breast cancer, but there is little evidence of genetic risk factors for benign tumors. AIM: To assess the combined genetic risk of developing mastopathy in women with FSHR (rs6165, rs6166) and ESR1 (rs9340799, rs2234693) gene variants. MATERIALS AND METHODS: The study included 87 infertile women (45 with concomitant fibrocystic mastopathy and 42 without mastopathy). RESULTS: For rs9340799 and rs2234693 variants of the ESR1 gene, we did not find any significant differences in the distribution of genotypes in infertile women with or without mastopathy. In patients with mastopathy, there was a reliable increase in the frequency of 307Ala/Ala and 680Ser/Ser genotypes of FSHR gene (χ2 = 6.39, p = 0.012, OR = 4.49 (1.48-13.65)) as compared to patients without mastopathy. In the presence of 307Thr/Thr and 680Asn/Asn genotypes of the FSHR gene, a 4.88-fold reduction of mastopathy risk (χ2 = 8.06, p = 0.005, OR = 0.21(0.07-0.59)) was observed. The frequency of the FSHR and the ESR1 genotypes combinations - 307Thr/Thr+680Asn/Asn+351AG+397TC was significantly decreased in patients with mastopathy. CONCLUSIONS: Our study did not find an association of ESR1 gene variants with the risk of developing of mastopathy in infertile women although heterozygous variants of the ESR1 gene enhanced the "protective" effect of FSHR gene variants and reduced the risk of mastopathy.

A variant of TP53 gene (rs 1625895, 13494g>A) is associated with neoplasm localization in patients with uterine leiomyoma.

BACKGROUND: Uterine leiomyoma (UL) is the most common benign neoplasm of the uterus. It is still unknown surely what exactly initiates transformation of the uterine myometrial cells into UL. AIM: To study the effect of the TP53 gene variants on the risk of development and clinical features of UL. MATERIALS AND METHODS: Case-control study was performed using molecular genetic analyses of variants rs1042522 (119 G>C) and rs1625895 (13494G>A) of TP53 gene in patients with UL and comparison group of healthy women. RESULTS: Investigated TP53 gene variants were not associated with the risk of UL development. The patients with the 13494GG genotype (rs1625895) had significantly more often subserous UL (р < 0.05). In patients with heterozygous variant of TP53 - 13494GA genotype (rs1625895) intramural UL was observed (р < 0.05). CONCLUSIONS: The rs1625895 (13494G>A) variant of TP53 gene was associated with UL localization. The identified dependence of the UL localization on the TP53 gene variant could be useful for personalized approach to treatment.

Prevalence of BRCA1 and BRCA2 genes promoter hypermethylation in breast cancer tissue.

BACKGROUND: Recent advances in the treatment of breast cancer (BC) have been related to the personalization of therapy. The methylation status of the promoter regions of tumor suppressor genes such as BRCA1 and BRCA2 is supposed to be useful as a prognostic factor in BC patients. AIM: To investigate the frequency of hypermethylation in the promoter regions of BRCA1 and BRCA2 genes in tumor tissue of BC patients, and the relation of hypermethylation to the clinical course of the disease. MATERIALS AND METHODS: Molecular genetic studies were performed on 50 BC tissue samples in order to determine the methylation status of the promoter regions of the BRCA1 and BRCA2 genes. RESULTS: Hypermethylation of the BRCA1 promoter region was detected in 34% of BC cases, hypermethylation of the BRCA2 promoter region - in 50% of cases, and hypermethylation of the promoter region of both genes - in 20% of cases. A significant increase in the incidence of hypermethylation of the BRCA2 promoter region was found in the group of patients older than 56 years, mainly in patients with triple-negative breast cancer and without family history of BC. CONCLUSIONS: The high frequency of hypermethylation in the promoter regions of BRCA1 and BRCA2 genes, as well as their co-methylation in tumor tissue of BC patients has been detected.

ESR1 GENE RELATED RISK IN THE DEVELOPMENT OF IDIOPATHIC INFERTILITY AND EARLY PREGNANCY LOSS IN MARRIED COUPLES.

The aim of the work was to assess genetic risk of reproductive disorders in married couples, conditioned by polymorphic variants A-351G and T-397C of ESR1gene. The study involved 412 persons - 206 married couples: 69 married couples with idiopathic infertility, lasting over 5 years, and 137 married couples with early reproductive losses in their past medical history. The data of population frequencies for the European population, obtained from the open database of 1000 Genomes project, were used as a comparison group. The polymorphic variants A-351G and T-397C of ESR1 gene were investigated using the method of polymerase chain reaction with subsequent analysis of the restriction fragment length polymorphisms. It was determined that the presence of genetic variant -351GG (log-additive model of inheritance) and the combination of genotypes -351GG/-397CC of ESR1 gene was associated with the increasing risk of developing male idiopathic infertility. The association of the polymorphic variant A-351G of ESR1 gene with the increasing risk of developing idiopathic infertility (log-additive model of inheritance) and early reproductive losses (over-dominant model of inheritance) was revealed in women from the examined married couples. Significant protective effects in terms of reproductive disorders in men were found for the combinations of genotypes -351AA/-397TT and -351AA/-397TC of ESR1 gene. The obtained results demonstrated new view about the ESR1 identical genetic mechanisms of developing idiopathic infertility and early pregnancy loss in couples. These determined specificities highlight the need of conducting genetic investigations of both ESR1 polymorphic variants in couples and the significance of searching for phenotypic manifestations of investigated reproductive disorders which occurred due to genetic variants.

POSITIVE EFFECT OF BETAINE-ARGININE SUPPLEMENT ON IMPROVED HYPERHOMOCYSTEINEMIA TREATMENT IN MARRIED COUPLES WITH REPRODUCTIVE DISORDERS.

Treatment of hereditary hyperhomocysteinemia and the achievement of optimal folate status is necessary for persons of reproductive age in order to increase live birth rate. Patients are usually advised to take folic acid, a key nutrient in homocysteine remethylation. The results of study showed risk factors for hyperhomocysteinemia development in investigated married couples: male gender, MTHFR, MTR1 genes variants, lower vitamin B12 blood serum and no additional intake of vitamin B12. Since MTHFR, MTR1 genes variants affect to decrease the efficiency of homocysteine metabolic transformations, to contribute also to endothelial dysfunction in one of patients group we used betargine combined with folic acids and vitamin B12 administration. Patients group with combined administration including betargine within 2 weeks, in comparison with the group without its supplement, had significantly decreased level of homocysteine in plasma, less than 12 µmol/l (81.03% and 50% of cases, respectively). Folic acid and vitamin B12 mean values in blood serum was significantly increased in patients after two week vitamins administration including betargin. Further research is needed to establish the duration of betaine-arginine intake until the target homocysteine level will be reached, as well as to estimate the durability of clinical effect achieved after consumption.

Genetic polymorphisms of the renin-angiotensin system in breast cancer patients.

BACKGROUND: Breast cancer (BC) is one of the most common cancer pathologies in women. Genetic polymorphism of genes of renin-angiotensin system (RAS) is considered to be associated with cancer development, in particular, with BC. AIM: To study the influence of polymorphic variants of genes coding for RAS components, on the risk of BC development in Ukrainian women. MATERIALS AND METHODS: In the study 131 patients with histologically proven diagnosis of BC of I and II stages were enrolled. The control group was composed from 102 women without cancer. Polymorphic variants of AGT, ACE, AT2R1 genes were studied with the use of PCR and PCR-RFLP methods. RESULTS: It has been revealed that the presence of 1166АС genotype of AT2R1 gene elevates the risk of BC development nearly 2-fold. The results of analysis for common group and subgroups distributed by age are different. For women from 18 to 35 years old the significant differences were not found. For women from 36 to 54 years old an increased risk of BC development is determined by the presence of D allele of АСЕ gene. Decreased risk of BC development was associated with the presence of combined genotypes ACE II/AGT 174TT and ACE II/AGT 235МТ. In women older than 54 years an increased risk of BC development was found to be related to the presence of genotypes 235ТT of AGT gene and 1166АС of AT2R1 gene. The presence of genotype combinations AGT 235ТТ/AGT 174ТМ and AGT 235ТТ/AT2R1 1166AA in women of this age subgroup also significantly increases the risk of BC development. CONCLUSION: These polymorphic gene variants and their associations may be considered as possible prognostic markers of BC development. The results of analysis are different in total cohort and in subgroups distributed by age.