STEREOTYPED CASES IN UKRAINIAN COHORT OF CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS DEPENDING ON THE IONIZING RADIATION EXPOSURE. / STEREOTYPNI VYPADKY V UKRAÏNS'KII KOGORTI KhVORYKh NA KhRONIChNU LIMFOTsYTARNU LEIKEMIIu ZALEZhNO VID VPLYVU IONIZUIuChOGO VYPROMINIuVANNIa.

OBJECTIVE: to analyze the stereotyped subsets in cohort of Ukrainian chronic lymphocytic leukemia (CLL) patients in general and depending on the ionizing radiation (IR) exposure. METHODS: Analysis was performed in the groups of 118 CLL patients irradiated due to the Chornobyl NPP accident (95 clean-up workers, 17 inhabitants of radionuclide contaminated areas, and 6 evacuees) and 294 IR non-exposed patients. The IGHV (immunoglobulin heavy chain variable region) gene mutational status, mutations of NOTCH1, TP53 and SF3B1 genes were studied by polymerase chain reaction followed by direct sequencing. Associations between clinical and molecular data of patients were analyzed with the SPSS software package, version 20.0. RESULTS: The incidence of stereotyped CLL cases in Ukrainian cohort was high (50.5 %) and comparable in IR-exposed and non-exposed patients. The ratio of major and minor clusters as well as the frequency of individual clusters was comparable with reported data with some exceptions: a low incidence of subset #2; absence of subset #8; high frequency of minor subset #V4|J4.5.6|18|5. The distinctive features of IR-exposed CLL patients found were:1) comparable frequency of stereotyped cases among mutated and unmutated (UM) IGHV genes cases (p = 0.557);2) lack of differences IGHV gene repertoires among stereotyped and heterogeneous cases (p = 0.508); 3) «heterogeneity¼ of stereotyped cases: all identified stereotyped clusters, with the exception of cluster #1, consisted of one case. Stereotyped cases with expression of UM IGHV clan I genes (except IGHV1-69 gene) were more susceptible to the appearance of NOTCH1 mutations. Patients of cluster #4 were younger, tended to have a longer time-to-treatment period and overall survival (OS) compared to subset #2. Patients of cluster #2 are more likely to have autoimmune hemolytic anemia (AIHA) and SF3F1 mutations. IGHV3-21 expression was associated with worse OS in univariate and multivariate analysis. AIHA was more common in patients with UM IGHV4-59 and IGHV3-11 genes. CONCLUSIONS: The revealed differences in distribution of stereotyped CLL cases in Ukrainian cohort are most likely to reflect variations in the genetic background, environmental factors (including IR exposure), and their interactions in different geographic areas.

EXPRESSION OF IMMUNOGLOBULIN SEQUENCES HOMOLOGOUS TO ANTI-SARS-CoV-2 ANTIBODIES AND HIV IN CHRONIC LYMPHOCYTIC LEUKEMIA CASES.

BACKGROUND: Identification of epitopes recognized by leukemic B cells could provide insights into the molecular mechanisms of B cell transformation in chronic lymphocytic leukemia (CLL). The aim of this paper was to compare nucleotide sequences of immunoglobulin heavy chain variable region (IGHV) genes in CLL with known sequences directed against antigens of different origins available in public databases. MATERIALS AND METHODS: Analysis was performed in the groups of 412 unselected CLL patients with productive IGHV gene using polymerase chain reaction followed by direct sequencing. RESULTS: Homology between CLL Ig sequences and antibodies directed against autoantigens was found in 12 patients (2.9%), homology between CLL Ig sequences and antiviral antibodies - in 35 patients (8.5%). Most of these sequences belonged to stereotypical clusters. Among the sequences that have homology to antiviral antibodies, the most prevalent were cases homologous with antibodies against HIV (14 cases, 3.4%) and SARS-CoV-2 antigens (10 cases, 2.4%). None of the patients in our cohort was HIV-infected and the study was conducted before the emergence of SARS-CoV-2 virus. CONCLUSIONS: Suggestions could be made about the possible impact of past infection of SARS-CoV-2 virus on the pathogenesis of CLL. In particular, an increase in the proportion of CLL cases with the expression of some stereotyped BCR and/or an increase of CLL risk in the long-term period after SARS-CoV-2 virus infection is not excluded. This assumption needs to be verified by epidemiological data.

THE EXPRESSION OF THE MAIN AND ALTERNATIVE TRANSCRIPT (SORL1 Delta2) OF THE SORL1 GENE IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS AFFECTED BY THE CHORNOBYL ACCIDENT. / DOSLIDZhENNIa OSNOVNOGO I ALTERNATYVNOGO TRANSKRYPTU (SORL1 Delta2) GENA SORL1 U KhVORYKh NA KhRONIChNU LIMFOTsYTARNU LEAKEMIIu, POSTRAZhDALYKh VNASLIDOK ChORNOBYLSKA KATASTROFY.

OBJECTIVE: to study clinical-hematological data and expression of the main and alternative transcripts of SORL1 genein chronic lymphocytic leukemia (CLL) patients affected by the Chornobyl catastrophe. METHODS: Analysis was performed in the main group of 34 CLL patients irradiated due to the Chornobyl NPP acci-dent (30 clean-up workers, and 4 evacuees) and in the control group of 27 non-irradiated CLL patients. Groups ofpatients were comparable by age, sex, stage of disease, mutational status of IGHV genes. Expression of the main andalternative transcripts of SORL1 gene was evaluated by Quantitative Real-time polymerase chain reaction (PCR). TheIGHV gene mutational status, TP53 and SF3B1 mutations were studied by PCR followed by direct sequencing. Data wereanalyzed with the SPSS software package, version 20.0. RESULTS: Relative expression level of the main transcript of SORL1 gene was low (mean 1.71 ± 0.55, median 0.57),did not correlate with the IGHV gene mutational status, TP53 and SF3B1 mutations, stage of disease. The expressionof B transcript was not detected, F transcript was expressed at a very low level in 9 patients. The average relativeexpression level of SORL1-Δ2 transcript was 14.1 ± 6.04 (median 3.48; range 0.01-90.51). The expression of SORL1-Δ2transcript above the median was more frequent among patients on C stage (p = 0.001), and in patients with unmu-tated IGHV genes was associated with an extremely negative course of CLL (median of overall survival 9 months vs61 months at low expression). Relative expression levels of the main and alternative transcripts of SORL1 gene inpatients of the main and the control groups did not differ. CONCLUSIONS: Our preliminary data suggest that increased expression of SORL1-Δ2 transcript in CLL patients withunmutated IGHV genes can be considered as a negative prognostic marker.

Association of lipoprotein lipase expression with TP53 gene polymorphisms in chronic lymphocytic leukemia cells.

BACKGROUND: Expression of lipoprotein lipase (LPL) correlates with unmutated (UM) status of the variable region of the heavy chain of immunoglobulin (IGHV) genes, but the expression level of LPL in UM chronic lymphocytic leukemia (CLL) cases varies significantly. AIM: To study the association of LPL expression with the genetic variants of the TP53 gene since both genes are involved in lipid metabolism. MATERIALS AND METHODS: Expression of LPL mRNA was measured in peripheral blood mononuclears of 45 CLL patients with UM IGHV genes by real-time quantitative reverse transcription polymerase chain reaction. Mutational status of IGHV genes and TP53 genotyping (rs1042522, rs1642785, rs17883323, rs2909430, rs145153611, rs113530090, rs12947788, rs12951053, and rs17878362) were performed by polymerase chain reaction amplification followed by direct sequencing. RESULTS: Observed CLL patients were divided on groups with low (11.17 ± 2.66) and high (275.48 ± 39.37) LPL expression. In CLL patients with UM IGHV genes and low LPL expression we found an increased frequency of rs1042522 G (p = 0.0036), rs1642785 C (p = 0.0001), and rs17878362A2 alleles (p = 0.0091). The possible functional significance of these changes is discussed. CONCLUSION: Some polymorphic variants of TP53 may be genetic modifiers for LPL expression level in CLL leukemic B-cells. Further research is required in a larger cohort to confirm these findings.

EXPRESSION OF LIPOPROTEIN LIPASE AND c-MYC ONCOGENE IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA AFFECTED BY THE CHORNOBYL ACCIDENT. / EKSPRESIIa GENA LIPOPROTEÏNLIPAZY I ONKOGENA c-MYC U KhVORYKh NA KhRONIChNU LIMFOTsYTARNU LEIKEMIIu, IaKI POSTRAZhDALY VNASLIDOK AVARIÏ NA ChORNOBYL'S'KII AES.

OBJECTIVE: to determine the association between the expression of lipoprotein lipase (LPL) and c-MYC genes inperipheral blood cells of chronic lymphocytic leukemia (CLL) patients affected by the Chornobyl catastrophedepending on the mutational status of IGHV genes. METHODS: Analysis was performed in the group of 69 CLL patients irradiated due to the Chornobyl NPP accident (58clean-up workers of 1986 year, 6 inhabitants of radionuclide contaminated areas, and 5 evacuees). The IGHV genemutational status was studied by polymerase chain reaction (PCR) followed by direct sequencing. LPL and c-MYCexpression was evaluated by Quantitative Real-time PCR. Data were analyzed with the SPSS software package, version 20.0. RESULTS: Relative LPL expression levels in CLL samples ranged from 0 to 1663.5 (mean 138.47 ± 30.69, median 26.1).A strong correlation between individual LPL expression levels and IGHV mutational status was found (r = 0.684;p < 0.0001). The average relative c-MYC expression level was 5.7 ± 0.87 (median 2.86; range 0-48.5). No association between c-MYC expression and IGHV mutational status was found. Among unmutated IGHV cases, a correlationbetween LPL and c-MYC gene expression levels was identified: r = 0.351; p = 0.013. CONCLUSIONS: Our data confirm the dominant concept that unmutated IGHV CLL cases are more sensitive to the actionof proliferative stimuli compared to mutated IGHV CLL cases. This is manifested by an increase in the expression ofa functionally significant LPL gene, is one for the strongest negative prognostic markers in CLL.

Analysis of immunoglobulin heavy variable chain rearrangement in chronic lymphocytic leukemia patients among Chornobyl clean-up workers.

BACKGROUND: A number of epidemiological studies have shown an elevated radiation-associated risk for chronic lymphocytic leukemia (CLL). The aim of the paper was to analyze immunoglobulin heavy variable chain (IGHV) rearrangement and IGHV usage in CLL cases associated with ionizing radiation (IR) exposure. MATERIALS AND METHODS: Samples of 76 clean-up workers of Chornobyl Nuclear Power Plant accident of 1986 (the main group) and 194 non-exposed patients (the control group) were analyzed. Two groups of CLL patients were comparable by gender (all patients were male), age, and place of residence (rural or urban). RESULTS: Some features of IR-associated CLL cases as compared to CLL cases in patients without history of IR exposure were revealed. Among unmutated IGHV sequences, IGHV1 genes were less commonly used (29.4% vs 48.6%; p = 0.018), while the frequency of IGHD6 genes was higher (23.5% vs 10%; p = 0.029). The unmutated IGHV sequences did not use IGHD3-16 gene (0% vs 7.9%, p = 0.038). Mutated IGHV sequences were less frequently expressed IGHV3 genes (44% vs 68.5%; p = 0.037) due low representation of IGHV3-21 (4% vs 11.1%) and IGHV3-23 (0% vs 11.1%) genes; did not use IGHD3-22 gene (0% vs 18.5%, p = 0.025); and have signs of positive selection in the HCDR regions (Σ = 0.5029 ± 0.155 vs -0.0539 ± 0.14; p = 0.013). CONCLUSIONS: The revealed differences in IGHV gene usage and B-cell receptor structure in the main and the control groups of CLL patients indirectly indicate a change in the spectrum of antigens associated with CLL under IR exposure. The possible antigenic drivers associated with CLL associated with IR exposure are discussed.

MYC copy number and mRNA expression in chronic lymphocytic leukemia patients exposed to ionizing radiation due to the Chornobyl NPP accident.

Some clinical and biological features indicating an unfavorable course of the disease were found in ionizing radiation (IR) - related chronic lymphocytic leukemia (CLL) patients. The MYC proto-oncogene is considered to contribute to CLL pathogenesis. Increased MYC copy number is associated with poor prognosis in CLL. AIM: To investigate the frequency of MYC gene copy number amplification in IR-exposed CLL patients and relate the findings to the MYC mRNA levels, the presence of unfavourable prognosis mutations (TP53, SF3B1, NOTCH1), and patient`s outcome. MATERIALS AND METHODS: The analysis of MYC copy number was carried out by real-time quantitative polymerase chain reaction (PCR) in 70 IR-exposed CLL patients. The MYC mRNA expression was measured by real-time quantitative reverse transcription PCR. RESULTS: Increased MYC gene copy number was present in 5.7% of cases. There was a statistically significant association between increased MYC copy number and increased MYC mRNA (p < 0.014). Additionally, somatic deletion in MYC locus was found in one patient. Most of patients (80%) with detected MYC aberrations were previously untreated, suggesting that these lesions might occur early in the course of the disease. The MYC aberrations were found mutually exclusive with high risk TP53 and SF3B1 mutations, while one case was identified, where MYC amplification and NOTCH1 mutation coincided simultaneously. Regarding clinical outcome, the MYC aberrations were associated with a shorter time to first treatment (3 vs 25 months, p = 0.008) as well as reduced overall survival (60 vs 139 months). CONCLUSION: Our data suggest that MYC aberrations might be an early event in IR-related CLL and contribute to aggressive disease development in the absence of high risk TP53 and SF3B1 mutations.

Analysis of LPL gene expression in patients with chronic lymphocytic leukemia.

AIM: The IGHV mutational status is one of the most important markers for chronic lymphocytic leukemia (CLL) prognostication. Lipoprotein lipase (LPL) gene expression was found to correlate with IGHV status and was suggested as its surrogate marker. Recent data reported that LPL expression might be influenced by pivotal signalling pathways in CLL. This study aimed to assess LPL gene expression in relation to key immunogenetic and molecular markers of CLL, including IGHV mutational status, B-cell receptor (BCR) stereotypy, TP53, NOTCH1, and SF3B1 gene mutations. Materials and Methods: Expression of LPL mRNA was measured in peripheral blood mononuclear cells of 73 CLL patients by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). IGHV, NOTCH1, TP53, and SF3B1 gene mutation analysis was performed by PCR amplification and direct sequencing. RESULTS: 44 of 73 (60%) CLL cases were categorized as LPL-positive based on the cut-off value established by ROC (receiver operating characteristic) curve analysis. LPL expression was significantly associated with IGHV mutation status (r = 0.684; p < 0.0001) and tended to correlate with presence of NOTCH1 gene mutations (p = 0.113). BCR stereotyped cases showed higher LPL expression values in comparison to unstereotyped cases in the LPL-positive group of patients (p = 0.041). LPL expression was associated with a shorter overall survival in the entire СLL group (median 107 vs 143, p = 0.048) as well as in Binet A patients, albeit with borderline significance (median 139 vs not reached, p = 0.086). CONCLUSION: LPL expression was found to be closely correlated with IGHV gene mutational status and overall survival, proving LPL as prognostic marker in CLL. Our results also indicate a possible relationship between aberrant expression of LPL and BCR- and NOTCH1-dependent signalling pathways.

GENDER DISPARITIES OF CIRCULATORY DISEASE PROGRESS IN THE CHORNOBYL ACCIDENT CLEAN-UP WORKERS AND PHOSPHODIESTERASE 4D GENE rs966221 POLYMORPHISM. / GENDERNI VIDMINNOSTI ROZVYTKU ZAKhVORIuVAN' SYSTEMY KROVOOBIGU V UChASNYKIV LIKVIDATsIÏ NASLIDKIV ChORNOBYL'S'KOÏ AVARIÏ I POLIMORFIZM rs966221 GENA FOSFODIESTERAZY 4D.

OBJECTIVE: Evaluation of the hypertensive disease (HD) and coronary heart disease (CHD) progress in the ChornobylNPP (ChNPP) accident clean-up workers (ACUW) and persons not exposed to ionizing radiation depending on gen-der and genotype of the phosphodiesterase 4D (PDE4D) gene rs966221 polymorphism. MATERIALS AND METHODS: There were male ACUW (ACUWm; n=515) and female ACUW (ACUWf; n=145) involved in thestudy since 2013 till 2018. Participation in the clean-up works took place in 1986-1987. The control group includ-ed male (CGm; n=162) and female (CGf; n=120) persons not exposed to ionizing radiation. All study subjects havehad neither signs nor symptoms of HD or CHD before the ChNPP accident. RESULTS: Review of the Kaplan-Meier survival tables indicated that according to median survival the HD emerged inACUWm and ACUWf in a younger age (47.5 ± 0.6 and 50.7 ± 0.7 years old, respectively) vs. CGm or CGf (54.9 ± 1.1 and54.4 ± 1.1 years, respectively). The same was true for CHD where the median values were (56.8 ± 0.5), (61.2 ± 0.8),(61.6 ± 1.0) and (64.2 ± 1.4) years respectively. Review of cumulative incidence of HD and CHD revealed no associ-ation of the PDE4D gene rs966221 polymorphism with the diseases of concern. The TT gene carrier state comparedto the CC or CT genes features an increased risk of myocardial infarction (MI) 2.9 times in ACUWm, 4-fold in CGm, and5.5 times in CGf (p < 0.05). No any gene carrier state was associated with MI in the ACUWf. Onset of menopause wasfollowed by an increase in HD incidence vs. males. CONCLUSIONS: The male and female ChNPP ACUW were developing HD and CAD at a younger age compared with cor-responding non-irradiated control. In male ACUW in comparison with female ACUW the cumulative morbidity ratefor MI was higher in any age range, whereas for CAD it was higher from 23 to 74 years, and for HD from 25 to 53 yearsof age. In male and female ACUW as well as in non-irradiated control the HD developed much earlier than CHD. Thecarrier state of TT genotype of PDE4D gene rs966221 polymorphism increases the risk of MI in males of all ages, inthe non-irradiated controls it is increased in 65 years for men and in 60 years for women. No data on association ofthe genotype of the described gene polymorphism with MI were found in female ACUW.

THE SPECTRUM OF TP53, SF3B1, AND NOTCH1 MUTATIONS IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS EXPOSED TO IONIZING RADIATION DUE TO THE CHORNOBYL NPP ACCIDENT. / SPEKTR MUTATsII GENIV TP53, SF3B1 ta NOTCH1 U KhVORYKh NA KhRONIChNU LIMFOTsYTARNU LEIKEMIIu, IaKI ZAZNALY VPLYVU IONIZUIuChOGO VYPROMINENNIa VNASLIDOK AVARIÏ NA ChORNOBYL'S'KII AES.

OBJECTIVE: to analyze TP53, NOTCH1 and SF3B1 mutations in chronic lymphocytic leukemia (CLL) patients, sufferersof Chornobyl NPP accident to clarify the possible relationship between ionizing radiation (IR) and CLL. METHODS: Mutations of TP53, NOTCH1, and SF3B1 genes were studied by direct sequencing in the main group of 106 CLLpatients exposed to IR due to Chornobyl NPP accident and in the control group of 130 IR non-exposed CLL patients. RESULTS: We found TP53 and SF3B1 mutations with similar incidence in both groups - 11.3 % and 10.0 % in the maingroup, and 12.7 % and 11.5 % in the control group, respectively. In contrast, the frequency of NOTCH1 mutationswas lower in IR-exposed patients (6.7 % vs 17.7 %; p = 0.012). TP53 mutations were seen with equal frequency amongmutated (11.1 %) and unmutated (11.8 %) immunoglobulin heavy-chain variable gene (IGHV) cases in IR-exposedCLL patients, while the tendency to prevalence of TP53 mutations in unmutated compared with mutated IGHV caseswas found in the control group (14.1 % and 5.6 %, correspondingly; p = 0.178). In IR-exposed group SF3B1 muta-tions were combined with mutations in TP53 almost in half of detected cases. In opposite, in the control group therewas mutual exclusivity between SF3B1 and TP53 lesions (p = 0.001). Among IR-exposed CLL patients we found two dif-ferent cases with identical rare mutation of TP53 gene - c.665C>T substitution (Pro222Leu). This substitution is verylikely to represent inherited TP53 mutation, which may influence CLL development under IR exposure. CONCLUSION: Our preliminary data suggest that TP53 abnormalities are involved in CLL development in subjectsexposed at the Chornobyl accident and also a possible connection between inherited sensitivity to ionizing radia-tion caused by mutation in TP53, radiation and CLL development.

Analysis of the 3'UTR region of the NOTCH1 gene in chronic lymphocytic leukemia patients.

Deregulation of NOTCH1-signalling pathway is common in chronic lymphocytic leukemia (CLL). The most of studies are focused on detection of the hotspot c.7541_7542delCT NOTCH1 mutations in exon 34, while studies of mutations in the 3'UTR region are rare. The aims of work were to evaluate the frequencies of mutations in the 3'UTR region of the NOTCH1 gene (9:136,495553-136,495994) in Ukrainian CLL patients, the distribution of rs3124591 genotypes located in that area, and association of NOTCH1 mutations with structure of B-cell receptor. MATERIALS AND METHODS: Detection of mutations in the 3'UTR region of the NOTCH1 was performed by direct sequencing in 87 previously untreated CLL patients (from the total group of 237 CLL patients) with unmutated immunoglobulin heavy-chain variable (UM IGHV) genes and without mutations in hotspot regions of TP53, SF3B1, and exon 34 of NOTCH1 genes. RESULTS: Mutations in the 3'UTR region of the NOTCH1 were revealed in three of 87 CLL patients (3.4%). Two cases with non-coding mutations were related to subset #1 of stereotyped B-cell receptors, and one case belonged to stereotyped subset #28a. Analysis with inclusion of 30 UM IGHV cases with previously detected c.7544_7545delCT mutations revealed that the frequency of UM IGHV genes of I phylogenetic clan (except IGHV1-69) was significantly increased, and the frequency of UM IGHV3 and IGHV4 genes, on the contrary, was reduced in NOTCH1-mutated cases comparing with NOTCH1-unmutated cases (p = 0.002) and the general group (p = 0.013). SNP rs3124591 did not affect the risk of CLL and survival parameters of the patients. At the same time, differences were found in the frequency of IGHV gene usage and in the structure of HCDR3 in carriers of individual genotypes. CONCLUSION: The frequency of NOTCH1 mutations in 3'UTR region was low. Our findings confirmed current data on the association between the structure of the B-cell receptor and the appearance of NOTCH1 mutations. Some features of HCDR3 structure were identified in carriers of TT and CC genotypes of rs3124591.

Influence of polymorphic variants of the SLC6A4 gene on the frequency of detection of depressive states in the group of the clean up workers of consequences of Chornobyl accident in the remote period after the Chornobyl catastrophe. / VPLYV POLIMORFNYKh VARIANTIV GENA SLC6A4 NA ChASTOTU VYIaVLENNIa DEPRESYVNYKh STANIV V GRUPI ULNA NA ChAES U VIDDALENOMU PERIODI PISLIa ChORNOBYL'S'KOÏ KATASTROFY.

Mental disorders of the victims are one of the important medical consequences of the Chornobyl accident. It is also known that in the implementation of the pathogenesis of depressive states a significant role belongs to the sero tonin transporter gene (SLC6A4). OBJECTIVE: To determine the effect of polymorphic variants of the SLC6A4 gene on the frequency of detection of depression in a group of clean up workers in the remote period after the Chornobyl catastrophe. METHODS: The study was conducted in a group of 59 victims of the Chornobyl NPP accident, divided into two groups (without depression and with depressive symptoms). The diagnosis of depressive disorders was based on a compre hensive assessment of the complaints of the surveyed, the clinical and psychopathological data, the values of the Zung Self Rating Depression Scale (SDS), the Brief Psychiatric Rating Scale (BPRS), the General Health Questionnaire (GHQ 28). DNA from the peripheral blood mononuclear cells was isolated, and the 5 HTTLPR polymorphism was determined by polymerase chain reaction (PCR). RESULTS: Depressive symptoms were more often found among reconvalescents of acute radiation sickness (ARS) than in the clean up workers without ARS: (p = 0.006). The tendencies of the association of the received dose of exter nal exposure with the number of points on the SDS scale (r = 0.284; p = 0.043), the sum of scores on the BPRS scale (r = 0.686; p = 0.001), depression (r = 0.323, p = 0.017) and its severity (r = 0.273; p = 0.051) were found. Among the examined clean up workers, in comparison with a large group of Europeans without mental disorders, an increase in the number of carriers of the genotype S/S SLC6A4 was found (p = 0.03). Only for the carriers of the S/S genotype, the reciprocal association between the development of depression and the age of the patient was found: r = 0.503 (p = 0.033), between the development of depression and the time from the ChNPP accident: r = 0.581 (p = 0.011), as well as positive correlation of development of depression with dose of irradiation: r = 0.515 (p = 0.025). Among people aged 55 and older, the development of depression was associated with a decrease in the frequency of high ly functional genotype LА/LА to 4.76% versus 31.25% in the absence of depressed symptoms (p = 0.042). In the group of younger patients, the distribution of genotypes did not differ depending on the signs of depression (p = 0.476). CONCLUSION: The pilot analysis of the distribution of genotypes of the SLC6A4 gene for polymorphisms of 5 HTTLPR and rs25531 in the clean up workers group showed the promise of further studies of the contribution of LА/LА і S/S genotypes to the development of depressive states in combination with the action of the radiation factor.

Distribution of rs2124594 genotypes in chronic lymphocytic leukemia patients depending on radiation anamnesis. / ROZPODIL GENOTYPIV ZA POLIMORFIZMOM rs2124594 U KhVORYKh NA KhRONIChNU LIMFOTsYTARNU LEY̆KEMIIu Z URAKhUVANNIaM RADIATsIY̆NOGO ANAMNEZU.

OBJECTIVE: to test the method of polymerase chain reaction with following fragments' length restriction to deter mine the rs2124594 polymorphism and to study its contribution in the development of chronic lymphocytic leukemia (CLL) in the post Chornobyl period. METHODS: Genotypes of rs2124594 were determined in 109 patients with CLL of B cell origin including 53 patients irradiated due to the Chornobyl NPP accident. Genotypes distribution among CLL patients was compared with healthy persons of European origin (the 1000 Genomes Project data set was used as a reference). RESULTS: Validity of the tested method was confirmed by direct sequencing. Associations between CLL risks and C allele (OR = 2.37; 95 % CI 1.50-3.73; р = 0.003), CLL risks and CT genotype (OR = 2.10; 95 % CI 1.38-3.21; р = 0.0012) were found. Distributions of rs2124594 genotypes in exposed and non exposed to ionizing radiation CLL patients did not differ. CONCLUSIONS: The association of single nucleotide polymorphisms across the 8q24 chromosome region (positioned at 127180736 and 127183014 near с MYC gene) with CLL risks was confirmed. Modified influence of ionizing radia tion on genetic susceptibility associated with rs2124594 was not found in this pilot study.

Detection of NOTCH1 c.7541_7542delCT mutation in chronic lymphocytic leukemia using conventional and real-time polymerase chain reaction.

UNLABELLED: To evaluate real-time polymerase chain reaction (PCR) assay system for detection of NOTCH1 c.7541_754delCT mutation in chronic lymphocytic leukemia (CLL) patients. MATERIAL AND METHODS: A total of 325 CLL patients were included in the study. Screening for NOTCH1 c.7544_7545delCT was performed using conventional PCR-based amplification refractory mutation system (ARMS) method. All 33 samples harboring c.7544_7545delCT allele and 5 negative cases as control were submitted to real-time PCR. RESULTS: Specificity and sensitivity of two PCR techniques were comparable. NOTCH1 c.7544_7545delCT mutation was found by ARMS in 10.1% of CLL patients, which is consistent with the data of other studies. However, the results of ARMS PCR in a minority of cases (2.15%) were doubtful and required reinvestigation. Real-time PCR, being less time-consuming, showed advantage in the assessment of the amplification's specificity (using the melting curve analysis). It also allows the quantitative assessment of NOTCH1-mutated clone. CONCLUSION: NOTCH1 c.7544_7545delCT mutation resulting in removal of the C-terminal PEST domain, deregulation of NOTCH1-dependent signaling pathways, has negative influence on prognosis of CLL and efficiency of therapy with anti-CD20 monoclonal antibodies. Real-time PCR allows the fast and reliable detection of c.7544_7545delCT mutation and can be used for the screening of this molecular lesion in CLL patients.

The distribution of TP53 gene polymorphisms in chronic lymphocytic leukemia patients, sufferers of Chornobyl nuclear power plant accident.

Previous analyses in a cohort of Chornobyl cleanup workers revealed significantly increased radiation-related risk for all leukemia types, including chronic lymphocytic leukemia (CLL). Numerous investigations emphasized the significance of genetic susceptibility to the radiation carcinogenesis. The aim of the work was to study the distribution of TP53 single nucleotide polymorphisms (SNPs) in CLL patients exposed to ionizing radiation (IR) due to Chornobyl nuclear power plant accident and estimate their impact on disease development. MATERIALS AND METHODS: The TP53 exonic and intronic SNPs were analyzed in 236 CLL patients by polymerase chain reaction and direct sequencing. The main group included 106 IR exposed CLL patients and thecontrol group was composed of 130 IR non-exposed CLL patients. RESULTS: Nineteen TP53 SNPs were found in the observed CLL cohort. No significant differences were found between the main and the control groups, but increased frequencies of T/T rs12947788 + G/G rs12951053 homozygotes and rs146340390 C/T variants were found among IR-exposed CLL patients compared with healthy Europeans (data from the 1000 Genomes Project). Rare nucleotide substitution rs146340390 (c.665C>T) was found only in the main group. These features were primarily typical for the most affected group of IR-exposed patients, namely, cleanup workers engaged in emergency works in the 2nd quarter of 1986. CONCLUSION: These preliminary findings don't contradict the assumption on possible influence of IR on CLL development via the p53-dependent pathway. This article is a part of a Special Issue entitled "The Chornobyl Nuclear Accident: Thirty Years After".

NOTCH1 mutations in chronic lymphocytic leukemia patients sufferers of Chornobyl NPP accident. / NOTCH1 MUTATsII U KhVORYKh NA KhRONIChNU LIMFOTsYTARNU LEYKEMIIu, IaKI POSTRAZhDALY VNASLIDOK AVARII NA ChORNOBYL'S'KIY AES.

UNLABELLED: Objective - to estimate the frequency of NOTCH1 mutations in chronic lymphocytic leukemia (CLL) patients, suffer ers of Chornobyl NPP accident, for elucidation of their impact in development of radiation associated forms of dis ease. METHODS: NOTCH1 mutations were determined by polymerase chain reaction followed by direct sequencing in 201 previously untreated patients with CLL of B cell origin: 88 CLL patients, sufferers of the Chornobyl NPP accident, and 113 CLL patients of the control group. RESULTS: Mutations of NOTCH1 were found in 13.4 % of observed patients, mostly in cases with unmutated heavy chain variable region (IGHV) genes (p = 0.001). Patients of the two groups were comparable by gender, age, stage at diagnosis, and mutational status of IGHV genes. But, the frequency of NOTCH1 mutations in the main group appeared to be lower in comparison with the control group (6.8 % vs 18.6 %; p = 0.015). Furthermore, if in the con trol group the number of NOTCH1 mutations increased in patients requiring first treatment compared with patients at diagnosis (p = 0.012), in the main group such differencies were non significant (p = 0.317). When clinical data of all observed groups of patients were analyzed, the presence of NOTCH1 mutations was associated with more advanced stage of disease, higher initial WBC count, bulky disease, short time to treatment period and progression free survival. CONCLUSION: Our data confirmed negative prognostic value of NOTCH1 mutations, but suggested to minimal impact of NOCTH1 mutations in CLL development under exposure to ionizing radiation.

TP53 codon 72 single nucleotide polymorphism in chronic lymphocytic leukemia.

UNLABELLED: Defects in the tumor suppressor gene TP53 are known to be important in chronic lymphocytic leukemia (CLL) and TP53 inactivation is associated with a particularly aggressive form of the disease. The single nucleotide polymorphism in the TP53 gene at codon 72 (rs1042522), results in amino acid substitution influencing apoptotic potential of TP53 protein. The aim of the study was to evaluate the association of the TP53 codon 72 polymorphism and incidence of TP53 mutations in CLL patients. METHODS: 261 CLL samples were analyzed by polymerase chain reaction and direct sequencing for TP53 mutations and single nucleotide polymorphism. RESULTS: The 72Pro/Pro genotype was associated with an increased incidence of TP53 mutations in previously treated patients (OR = 2.503; 95% CI 1.142-5.487; р = 0.001). CONCLUSION: This study revealed that the TP53 codon 72 polymorphism may be used as a risk factor for incidence of TP53 mutations in CLL.

Gene polymorphisms of p53-mediated apoptosis in chronic lymphocytic leukemia patients: features of distribution depending on radiation factor in anamnesis. / Polimorfizmy geniv r53-oposeredkovanogo apoptozu u hvoryh na hronichnu limfocytarnu lejkemiju: osoblyvosti rozpodilu zalezhno vid radiacijnogo chynnyka v anamnezi.

Objective. To set of p53-mediated apoptosis gene polymorphisms (TP53 codon 72 Arg/Pro, р21 codon 31 Ser/Arg, MDM2 SNP309) for the occurrence of CLL in patients who were exposed to ionizing radiation (IR) from the Chornobyl accident. Methods. Polymorphisms of p53-mediated apoptosis were determined in 320 patients with CLL of B-cell origin: 107 irradiated by the Chornobyl accident patients, 213 patients with CLL who had no history of exposure to IR, 73 individuals without a cancer and hematologic diseases that were affected by the Chornobyl disaster and 72 residents of Kyiv without affecting by IR in anamnesis. Determination of polymorphisms of p53-mediated apoptosis was performed by polymerase chain reaction followed by restriction. Results. The distribution of genotypes in patients with CLL did not differ from controls, except for reduced the frequency of homozygotes Arg/Arg TP53 among patients with CLL (p = 0.01). Compared with non-irradiated CLL patients in the subgroup of patients affected by the accident, an increase in the frequency of polymorphic alleles of the gene р21 (p = 0.033) was found, especially in combination with Arg allels (genotypes Arg/Arg and Arg/Pro) of TP53 gene and genotype TT SNP309 of MDM2 gene (p = 0.009). Conclusion. Preliminary studies indicated the likely contribution of rs1801270 polymorphism of the gene р21 in the pathogenesis of CLL in patients who had been exposed to IR. The effects of SNPs rs1042522 of TP53 gene and SNP309 of MDM2 gene on the risk of CLL in the Chornobyl accident sufferers were not revealed.

IGHV gene rearrangements as outcome predictors for CLL patients: experience of Ukrainian group.

Important characteristics of chronic lymphocytic leukaemia (CLL) cells are biased immunoglobulin variable heavy chain (IGHV) gene repertoire and expression of stereotyped B-cell receptors (BCRs); however, their prognostic value (in contrast to the impact of IGHV gene mutational status) is less clear. To evaluate the impact of separate IGHV gene usage and expression of stereotyped BCRs in CLL prognosis. Clinical data and IGHV gene configuration were analysed in 319 consecutive patients with CLL. We found that the majority of clinical parameters of patients were defined by IGHV mutational status. Our data also provided new evidence supporting the prognostic relevance of separate IGHV genes or stereotyped BCR in CLL, namely: (a) a restricted non-mutated (UM) IGHV gene repertoire in CLL patients with autoimmune haemolytic anaemia (AIHA) (more frequent expression of UM IGHV1-69, IGHV3-11 and IGHV4-59 genes, P = 0.001), a shorter period of AIHA development for expressors of these genes (P = 0.001) and a tendency towards expression of a stereotypic HCDR3 (P = 0.029), (b) a high incidence of second solid tumour development in IGHV3-21-expressing patients (P = 0.005) and (c) differences in overall survival (OS) of UM CLL patients depending on the BCR structure. Further research of specific IGHV gene usage and subsets of stereotyped BCRs in CLL may be helpful in more precise prediction of CLL prognosis in individual patients.

IGHV3-21 gene expression in patients with B-cell chronic lymphocytic leukemia in Ukraine.

UNLABELLED: THE AIM of the study was to evaluate the frequency of IGHV3-21 gene usage and its clinical significance for patients with B-cell chronic lymphocytic leukemia (CLL) in Ukraine. PATIENTS AND METHODS: Immunoglobulin variable heavy chain (IGHV) gene repertoire was studied in 189 CLL patients using reverse transcribed polymerase chain reaction and direct sequence of amplified products. RESULTS: IGHV3-21 gene expression was found in 11 cases (5.8%), and its frequency was intermediate between Scandinavian (11.7%) and Mediterranean CLL (2.9%) cohorts. The most of cases (9 of 11) belonged to subset with heterogeneous HCDR3 (heteroHCDR3 subset), and only 2 cases--to subset with classical short ARDANGMDV motif (homHCDR3 subset). Six IGHV3-21 cases were mutated and 5 cases were unmutated. All unmutated cases (all were from heteroHCDR3 subset) had similarity of their HCDR3s with previously published sequences. The differences in overall (OS), progression-free (PFS) and treatment-free survival (TFS) for IGHV3-21 positive patients in comparison with CLL patients expressing the other IGHV genes were statistically insignificant. These survival parameters were comparable also for CLL patients with mutated IGHV3-21 gene usage and expression the others mutated IGHV genes. But remarkable feature of IGHV3-21 expressing patients was high incidence of solid tumors. They have developed in 4 IGHV3-21 positive cases (36.4%) and in 10 cases with expression of the others IGHV genes (5.6%, p=0.0002). Furthermore, in small group of 6 patients with mutated IGHV3-21 gene expression, 3 patients had solid tumors and one underwent Richter transformation. Unmutated IGHV3-21 gene expressed patients had worse OS and PFS in comparison with CLL patients that expressed the others unmutated IGHV genes. CONCLUSION: Presented data are in agreement with the opinion about negative prognostic significance of IGHV3-21 gene expression regardless its mutation status. IGHV3-21 expression was associated with development of secondary solid tumors. Revealed high level of homology in heteroHDR3s subset might suggest about possible antigenic influence also, in addition to homHCDR3 subset that was proposed earlier.