Detection of notch1 c.7544_7545deICT mutation in chronic lymphocytic leukemia using conventional and real-time polymerase chain reaction

Aim: 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

TP53 codon 72 single nucleotide polymorphism in chronic lymphocytic leukemia

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. Key Words: chronic lymphocytic leukemia, TP53 mutations, single nucleotide polymorphism

The musculoskeletal system diseases in pregnant women with high infection risk and the single nucleotide rs1544410 polymorphism of the calcitriol receptor gene

Background. The problem of vitamin D (VD) deficiency in the population, in general, and in pregnant women, in particular, and related diseases, including the musculoskeletal system, remains one of the most widespread medical and social problems of our time. The purpose was to determine the frequency of musculoskeletal diseases in pregnant females at high infection risk (HIR) with impaired vitamin D status and single-nucleotide Bsml polymorphism of its receptor gene. Materials and methods. Fifty-six pregnant women (main group) with HIR and 40 healthy pregnant women (control group) had been examined. The level of 25-hydroxyvitamin D (25(OH)D) in blood was determined by the enzyme-linked immunosorbent assay, and real-time polymerase chain reaction was used to detect the mutant version of Bsml (rs1544410) polymorphism of the gene that is encoding vitamin D receptors (VDR). Statistical processing of the results was done using the resource www.socscistatistics.com. Results. HIR was due to the presence of chronic kidney diseases, carriers of pathogens of the TORCH group of infections and conditionally pathogenic microflora in the urogenital tract. The level of 25(OH)D was lower than the generally accepted optimal level in 76.8 and 15 % of pregnant women, in the main and control groups, respectively (F = 0.03; p = 0.0001). Carriers of the heterozygous genotype A/G were 67.7 % of pregnant women with HIR compared to 35 % of the control group (odd ratio (OR) = 3.95; 95% confidence interval (CI): 2.19–7.1; χ2 = 20.88, p = 0.00001), and the G/G genotype was inherent in 19.6 and 47.5 % of women, respectively (OR = 0.27; 95% CI 0.15–0.51;  p = 0.00006). A third of pregnant women from the main group had a history of musculoskeletal diseases (32.14 %) versus 12.5 % in control group (OR = 3.15; 95% CI: 1.54–6.46); 71.4 % of pregnant women with HIR were carriers of A/G genotype (OR = 9.79; 95% CI: 5.10–18.82). Conclusions. The share of vitamin D deficiency/insufficiency in pregnant women with HIR is almost 77 %. The general somatic history of these women is characterized by a high frequency of the kidney diseases (37.5 %) and musculoskeletal diseases (32.1 %). Two-thirds of pregnant women with HIR, as well as with musculoskeletal diseases, are carriers of the heterozygous Bsml of polymorphic genotype A/G of the VDR gene, which probably causes a higher risk of the development of pathology in conditions of calcitriol deficiency. Studying VD status, the genetic personification of disease risks, and correction of modified factors in time, in particular, VD deficiency is seen as a promising direction for improving perinatal outcomes and the quality of life of pregnant women in general, but further research is required

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. Key Words: NOTCH1 mutations, 3′UTR region of the NOTCH1, rs3124591, IGHV genes

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. Key Words: NOTCH1 mutations, 3′UTR region of the NOTCH1, rs3124591, IGHV genes