Allele-specific Real-time Polymerase Chain Reaction Research Articles

It was not possible to detect BRAF V600E mutation in circulating cell-free DNA from patients with ameloblastoma: A diagnostic accuracy study.

The objective of this study is to evaluate the diagnostic accuracy of plasma-based liquid biopsy for the detection of the BRAF V600E mutation in circulating cell-free DNA from patients with ameloblastoma. This is a prospective diagnostic accuracy study conducted based on the Standards for Reporting Diagnostic Accuracy recommendations. The index test was the plasma-based liquid biopsy, whereas the reference standard was the conventional tissue biopsy. The target condition was the detection of BRAF V600E mutation. The study population consisted of individuals with ameloblastoma recruited from three tertiary hospitals from Brazil. A negative control group composed of three individuals with confirmed wild-type BRAF lesions were included. The participants underwent plasma circulating cell-free DNA and tumor tissue DNA isolation, and both were submitted to using competitive allele-specific TaqMan™ real-time polymerase chain reaction technology mutation detection assays. Sensitivity and specificity measures and positive and negative predictive values were calculated. Twelve patients with conventional ameloblastoma were included. BRAF V600E mutation was detected in 11/12 (91.66%) ameloblastoma tissue samples. However, the mutation was not detected in any of the plasma-based liquid biopsy circulating cell-free DNA samples in both ameloblastomas and negative control group. The sensitivity and specificity of plasma-based liquid biopsy for the detection of the BRAF V600E mutation in circulating cell-free DNA was 0.0 and 1.0, respectively. The agreement between index test and reference standard results was 26.66%. Plasma-based liquid biopsy does not seem to be an accurate method for the detection of the BRAF V600E mutation in circulating circulating cell-free DNA from patients with ameloblastoma, regardless of tumor size, anatomic location, recurrence status, and other clinicopathological features.

PlentiPlex™ MYD88 Waldenström lymphoma qPCR assay: A highly sensitive method for detection of MYD88 L265P mutation.

Agarose gel-based conventional and real-time allele-specific polymerase chain reaction (AS-PCR) assays are currently used for sensitive detection and quantification of MYD88 L265P mutation. Visual inspection of an agarose gel can often be ambiguous. We propose a new allele-specific quantification PCR (AS-qPCR) assay, PlentiPlex™ MYD88 Waldenström lymphoma qPCR assay, that uses Intercalating Nucleic Acid (INA®) technology for increased affinity and specificity. This study compares PlentiPlex™ MYD88 Waldenström lymphoma qPCR assay with conventional AS-PCR. We included a total of 102 peripheral and bone marrow blood samples from 94 patients with a lymphoproliferative disorder. Droplet digital PCR (ddPCR) was used as a third method in case of discrepancy. A positive percent agreement of 100% (95% CI 0.92-1.0) and a negative percent agreement of 98% (95% CI 0.90-1.0) were found between the conventional AS-PCR and the AS-qPCR methods. Including the ddPCR results to validate the discrepant cases, the sensitivity and specificity of PlentiPlex™ MYD88 Waldenström lymphoma qPCR Assay were 1.0 (95% CI 0.97-1.0) and 1.0 (95% CI 0.96-1.0), respectively. Our data demonstrate that PlentiPlex™ MYD88 Waldenström lymphoma qPCR assay is a fast, highly sensitive, and specific method for the detection of MYD88 L265P compared with conventional AS-PCR.

Expression of JAK2 V617F Mutation in BCR - ABL Negative Myeloproliferative Neoplasms

OBJECTIVE: To detect the presence of JAK2V617F mutation in BCR - ABL negative myeloproliferative neoplasms and to stratify high risk patients for targeted therapy.PLACE AND DURATION OF STUDY: Department of Haematology, Armed forces institute of Pathology (AFIP), Rawalpindi, from Dec 2023 to Oct 2023.STUDY DESIGN: Descriptive cross sectional studyMETHODOLOGY: A total of 40 consecutive patients of BCR - ABL 1 negative myeloproliferative disorders were included in the study after being diagnosed according to WHO defined haematological criteria for each disorder and performing bone marrow examination under aseptic measures. Informed consent and ethical approval were obtained from each individual. All patients had their blood samples examined for the G-T point mutation (V617F) in the JAK2 gene located on chromosome 9, utilizing allele-specific real-time qualitative polymerase chain reaction (RT-qPCR). Results were analyzed by using SPSS version 22.RESULTS: Out of total 40 patients included in our study 21(52.5%) had Polycythemia Vera (PV), 15 (37.5%) had Primary myelofibrosis (PMF) and 4 (10%) had Essential thrombocythemia (ET). JAK2 mutation was found in 20 (95.2%) patients of PV, 9 (60%) patients of PMF and 2 (50%) patients of ET with a significant p-value of ˂ 0.001.CONCLUSIONS: JAK2 V617F is a useful clinical marker for establishing diagnosis and separating high risk patients for specific targeted therapy.

Association of <I>TNFAIP3</I> (rs10499194) and <I>TNF-α</I> (rs1800629) gene polymorphisms with susceptibility to systemic lupus erythematosus with juvenile onset and its clinical phenotypes in the Russian pediatric population

Numerous recent studies have shown that TNFAIP3 and TNF-α gene polymorphisms are associated with susceptibility to certain autoimmune and inflammatory diseases, including systemic lupus erythematosus (SLE), systemic scleroderma, rheumatoid arthritis, psoriasis, etc. However, the results of studies on associations between these polymorphisms and the risk of developing SLE in children are ambiguous and few in number.Objective: to test the hypothesis of a possible association between the rs10499194 polymorphism of the TNFA1P3 gene and the rs1800629 polymorphism of the TNF-α gene with susceptibility to juvenile SLE (jSLE) and its clinical phenotypes in the Russian pediatric population.Material and methods. Both polymorphisms were studied by allele-specific real-time polymerase chain reaction in 63 children (15 boys and 48 girls) with a confirmed diagnosis of jSLE, whose mean age was 12.3±3.2 years (3–17 years), and the mean duration of the disease was 4.1±2.4 years. Data on the frequency of genotypes and alleles of the corresponding TNFA1P3 and TNF-α gene polymorphisms in 309 healthy unrelated blood donors over the age of 18 years (20–45 years) were used as controls.Results and discussion. The study showed that the frequency of the rs10499194T mutant allele of the TNFA1P3 gene in patients with jSLE was significantly lower compared to the control (20.6 and 30.7%; p=0.023), and its carriage slightly reduced the risk of developing SLE (odds ratio, OR 0.58; 95% confidence interval, CI 0.32–1.05, p=0.053). The frequency of the rs1800629A mutant allele of the TNF-α gene was slightly higher in jSLE compared with controls (38.1 and 26.2%, respectively; p=0.056), and its carriage slightly increased the risk of developing SLE (OR 1.73; 95% CI 0.93–3.16; p=0.056). An analysis of the frequency distribution of the rs10499194 genotypes in groups of patients with and without arthritis revealed significant differences (p=0.003). Carrying genotypes with the mutant T allele (CT+TT genotypes) in jSLE significantly reduced the risk of developing of arthritis (p=0.003). At the same time, the risk of arthritis in carriers of at least one C allele was 3.76 times higher than in carriers of the other allele (p=0.006). No relationship was found between the rs1800629 TNF-α gene polymorphism and the clinical phenotypes of jSLE.Conclusion. The rs10499194T mutant allele statistically significant reduces the risk of arthritis development as one of the clinical manifestations of jSLE, and the rs1800629A mutant allele of the TNF-α gene is associated with a tendency to increase the risk of jSLE.

Allele-Specific PCR for PIK3CA Mutation Detection Using Phosphoryl Guanidine Modified Primers.

Phosphoryl guanidine (PG) is the novel uncharged modification of internucleotide phosphates of oligonucleotides. Incorporating PG modification into PCR primers leads to increased discrimination between wild-type and mutated DNA, providing extraordinary detection limits in an allele-specific real-time polymerase chain reaction (AS-PCR). Herein, we used PG-modification to improve the specificity of AS primers with unfavorable Pyr/Pur primer's 3'-end mismatch in the template/primer complex. Two mutations of the PIK3CA gene (E542K, E545K) were chosen to validate the advantages of the PG modification. Several primers with PG modifications were synthesized for each mutation and assessed using AS-PCR with the plasmid controls and DNA obtained from formalin-fixed paraffin-embedded (FFPE) tissues. The assay allows the detection of 0.5% of mutated DNA on the wild-type DNA plasmid template's background with good specificity. Compared with ddPCR, the primers with PG-modification demonstrated 100% specificity and 100% sensitivity on the DNA from FFPE with mutation presence higher than 0.5%. Our results indicate the high potential of PG-modified primers for point mutation detection. The main principle of the developed methodology can be used to improve the specificity of primers regardless of sequences.

PD-L1 expression in rare and aggressive thyroid cancers: A preliminary investigation for a role of immunotherapy.

Programmed cell death ligand-1 (PD-L1) immunoexpression status determines the response to immunotherapy in many cancers. Limited data exist on PD-L1 status in aggressive thyroid tumors. We investigated PD-L1 expression across thyroid cancers and correlated it with their molecular profile. Sixty-five cases of differentiated thyroid carcinoma, poorly differentiated thyroid carcinoma (PDTC), and anaplastic thyroid carcinoma (ATC) were assessed for PD-L1 expression (clone SP263, VENTANA). The differentiated cases encompassed the aggressive hobnail and tall cell subtypes of papillary thyroid carcinoma (PTC) besides classical PTC and follicular thyroid carcinoma (FTC). Ten nodular goiters (NG) were also evaluated. Tumor proportion score (TPS) and H-score were calculated. BRAFV600E and H-/K-/N-RAS were assessed using allele-specific real-time polymerase chain reaction (PCR). Fisher's exact and Kruskal-Wallis tests were used to investigate the associations between the categorical variables and compare PD-L1 scores with the mutation status. Most PTC (87%) and ATC (73%) cases were PD-L1 positive (TPS ≥1%), with significantly higher positivity rates than NG (20%). TPS >50% was seen in 60% ATC and 7% PTC cases. The median TPS and H-score of ATC were 56 (0-96.6) and 168 (0-275), respectively, and of PTC were 9.6 (4-16.8) and 17.8 (6.6-38.6), respectively. The scores were similar across the PTC subtypes. Only one case each of FTC and PDTC was PD-L1 positive. PD-L1 expression correlated significantly with BRAFV600E, but not with RAS mutation. ATC showed intense and diffuse PD-L1 staining. Although most PTCs were PD-L1 positive, the expression was weaker and patchy, irrespective of the histological subtype. Results of this pilot study indicate that ATC is most likely to respond to immunotherapy. PTC, FTC, and PDTC may be less amenable to immunotherapy. PD-L1 expression correlated significantly with BRAFV600E, allowing for combined targeted therapy.

A Review of Mitochondrial SNP Determination using Allele-Specific PCR in Forensic Identification

Allele-specific polymerase chain reaction (AS-PCR) is a rapid and cost-effective single nucleotide polymorphism (SNP) genotyping method compared to multiplex or real-time PCR. The SNPs occurring in mitochondrial DNA (mtSNPs) is the most abundant polymorphism in humans and can be used in human identification involving mass fatality incidents. Nevertheless, the application of AS-PCR has yet to be widely applicable in forensic investigations because this method requires further development due to its limitations. This review aims to relate the applications of AS-PCR, the combination of AS-PCR with other PCR techniques and improvements imposed on this technique in past studies. A narrative literature review was carried out over 70 studies related to the mtDNA, SNP, AS-PCR, and forensic DNA. The findings suggested that AS-PCR with new adaptations in technology such as incorporating additional mismatch, choosing allele-specific primer with either common forward or reverse, gel free real-time AS-PCR analysis, and usage of fluorescence probes can improve the specificity of SNP genotyping in forensic applications. This review concludes that AS-PCR and its combination with other improvised techniques can be applied in human identification to successfully analyze degraded or low quantity samples. Nevertheless, development of more SNP loci is required to implement AS-PCR in human identification to match the current gold standard STR typing method.

Expression of transcription, growth factors, steroid hormone receptors, LC3B in papillary thyroid cancer tissue, association with prognosis and risk of recurrence

Introduction. Biological characteristics of the tumor play a major role in it’s development and progression. Currently, using the molecular markers aimed at resolving the problems in clinical oncology is becoming more important, including thyroid carcinomas. Heterogeneous contradictory data had been accumulated to date showing the ability of tumors genetic and biological parameters to predict the diseases outcome.Aim. To investigate prognostic value of transcription, growth factors, components of AKT / mTOR signaling pathway and autophagy protein LC3B in patients with papillary thyroid cancer in relation to recurrences and overall survival.Materials and methods. The study included 65 patients with T1–4N0–1M0 papillary thyroid cancer. According to the criteria of the American Thyroid Association (ATA) (2015), patients were divided into groups of patients with high, low and intermediate risk. 30 patients were classified as low risk, 23 as intermediate risk, and 12 as high risk. The BRAFV600 mutation was identified in 18 samples. The expression of transcription factors (p65 and p50 subunits of nuclear factor kappa B (NF-κB p65, NF-κB p50), hypoxia-inducible factor 1 (HIF-1), hypoxia-inducible factor 2 (HIF-2), growth factors (vascular endothelial growth factor (VEGF), receptor VEGF (VEGF-2), carbonic anhydrases of type 9 (CAIX)), AKT, c-RAF, GSK- 3β, p70S6, mammalian target of rapamycin (m-TOR), PDK, PTEN, 4E-BP1 in the tumor was assessed by real-time polymerase chain reaction (PCR). The BRAFV600 mutation was investigated using real-time allele-specific PCR. The content of the LC3B protein was examined using the Western Blot method.Results. As a result of the study, there is an increase in c-RAF expression with an increase in risk from low to high, which was accompanied by a decrease in 4E-BP1 expression. c-RAF mRNA levels were increased 3.0- and 2.8‑fold in the intermediate and high-risk groups, respectively, compared to low risk patients. There is a change in the expression of Brn-3α depending on the relapse risk. The maximum mRNA levels were found in patients with intermediate risk, where the figure was 4.3 and 6.2 times higher than in patients with low and high risk, respectively. An increase in LC3B expression by 56.0 and 28.0 times was shown in the tumor tissue of patients with intermediate risk compared with patients with low and high risk. This fact corresponds with an increasing content of the protein itself, which was higher in patients with intermediate risk. Patients with a negative BRAF gene status had an intermediate and high risk of tumor recurrence. The prognostic significance of the estrogen receptor β (ER-β) and NF-κB p50 expression level had been revealed in relation with relapse-free and overall survival of patients with papillary thyroid cancer.Conclusion. As a result of the study, additional molecular markers were found in order to for predict the tumors recurrence risk. The study showed the significance of ERβ and NF-κB p50 expression levels for predicting disease outcomes.

Polymorphisms of the <i>TPMT</i>, <i>NUDT15</i> genes and 6-mercaptopurine toxicity profile in adult patients with Ph-negative acute lymphoblastic leukemia/lymphomas on the ALL-2016 protocol

Background. 6-mercaptopurine (6-MP) is a drug that is included in the treatment protocols for children and adults with acute lymphoblastic leukemias/lymphomas (ALL/LBL). It is known that individual differences in 6-MP tolerance can be explained by the TPMT and NUDT15 polymorphisms.Aim. To determine 6-MP toxicity profile in adult patients with Ph-negative ALL/LBL treated by ALL-2016 protocol, depending on the TPMT and NUDT15 polymorphisms.Materials and methods. The study included 54 adult patients with Ph-negative ALL/LBL (40 male and 14 female). The median age was 31 (18-51) years. T-ALL/LBL was diagnosed in 29 patients, B-ALL/LBL - in 22, acute leukemia with a mixed immunophenotype - in 3. All patients received treatment according to the multicenter study ALL-2016 (ClinicalTrials.gov, NCT03462095). polymorphisms in NUDT15 (*2, *3) and TPMT (*2, *3A, *3B, *3C) genes were detected using the allele-specific real-time polymerase chain reaction. Genomic DNA was extracted from patients peripheral blood samples. On the induction and consolidation therapy by the protocol, the received and proper 6-MP doses were calculated for all the patients. Drug toxicity was evaluated based on clinical and laboratory data.Results. TPMT and NUDT15 polymorphisms were detected in 11 (20 %) patients, more often in B-ALL - 7 (32 %) of 22 (p <0.05). A lower dose of 6-MP was received by patients with TPMT, NUDT15 polymorphisms only at consolidation IV (p = 0.01). we didn't find a correlation between the 6-MP toxicity and the polymorphisms in our patients (p >0.05).Conclusion. There were no differences in the received dose of 6-MP and the incidence of toxicity in adult patients between Ph-negative ALL/LBL with or without TPMT and NUDT15 polymorphisms treated according to ALL-2016 protocol (p >0.05). further studies including evaluation of 6-MP metabolites concentrations are required for a more complete understanding of the metabolism of this drug.

Frequency of PIK3CA mutations in different subsites of head and neck squamous cell carcinoma in southern Thailand.

BackgroundPhosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutations have been reported in many cancers, including head and neck squamous cell carcinoma (HNSCC). The frequency of these mutations varies among tumor locations and might be relevant to treatment outcomes among HNSCC. In this study, we examined the frequency of PIK3CA mutations in the different subsites of HNSCC.MethodsNinety-six fresh biopsy specimens were investigated for mutations in PIK3CA exons 4, 9, and 20 using allele-specific real-time polymerase chain reaction. Patient characteristics and survival were analyzed and compared between specimens with or without PIK3CA mutations.ResultsThe study included primary tumors originating from the oral cavity (n = 63), hypopharynx (n = 23), and oropharynx (n = 10). We identified mutations in 10.4% of patients (10 of 96 specimens). The overall mutational frequency was 17.4% (4/23) and 9.5% (6/63) in the hypopharynx and oral cavity, respectively. No patients with oropharyngeal carcinoma had mutations. Among the 10 mutant specimens, five were missense mutations (exon 9 [E545K] in two samples and exon 20 [H1047R] in three samples) and five were silent mutations in exon 20 (T1025T). Mutations were not found in exon 4. Among 84 patients with available clinical data, we found no significant differences in clinical characteristics and survival based on the presence or absence of PIK-3CA mutations.ConclusionsThe results indicate that PIK3CA mutations are involved in HNSCC carcinogenesis, and the hypopharynx should be considered a primary site of interest for future studies, particularly in Southeast Asian populations.

Involvement of polymorphisms of TNF, CRP and SAA1 genes in the predisposition to the development of ankylosing spondylitis and its clinical manifestations

Objective. To study the involvement of the rs1800629 G/A polymorphisms of the TNF-α gene, rs1205C/T of the CRP gene, and rs12218T/C of the SAA1 gene in the predisposition to ankylosing spondylitis (AS) and their role in the formation of clinical phenotypes of AS.Materials and methods. 122 patients with AS were included in the study. All patients had a diagnosis of AS based on the modified New York criteria. The presence of the HLA-B27 antigen was detected in 109 (89.3%) patients, the presence of peripheral arthritis – in 71 (58.2%), enthesitis – in 92 (75.4%), coxitis – in 82 (67.2%) patients. All patients had a high degree of activity with an average BASDAI index of 5.6±1.2. The control group consisted of 142 healthy blood donors. Polymorphisms were studied using allele-specific real-time polymerase chain reaction (RT-PCR).Results. Significant differences were found in the frequencies of genotypes and alleles of the -308G/A polymorphism of the TNF gene and the frequencies of alleles of the rs12218 T/C polymorphism of the SAA1 gene between patients and the control group (p=0.01, p=0.01 and p=0.03 respectively). Logistic regression analysis showed that the presence of at least one -308A allele in the patient’s genotype reduced the risk of developing AS by 4.4 times compared with the GG genotype (p=0.006). In carriers of the GA genotype, the probability of a predisposition to the development of enthesitis was 2.2 times lower than in carriers of the GG genotype (p=0.01). The relationship between the rs1205 polymorphism of the CRP gene and the predisposition to peripheral arthritis has been established. Carriage of the rs1205T allele doubled the susceptibility to arthritis compared with the rs1205C allele (p=0.013). Carriage of at least one rs12218C allele of the SAA1 gene doubled the susceptibility to AS (p=0.018).Conclusion. The data obtained confirm the involvement of polymorphisms rs1800629 of the TNF gene, rs1205 of the CRP gene, and rs12218 of the SAA1 gene in the predisposition to AS. TNF gene polymorphism is associated with the formation of the clinical phenotype of enthesitis, and CRP gene polymorphism is associated with a predisposition to peripheral arthritis.

Use of sewage surveillance for COVID-19 to guide public health response: A case study in Hong Kong

Sewage surveillance could help develop proactive response to the Coronavirus Disease 2019 (COVID-19) pandemic, but currently there are limited reports about examples in practical exercises. Here, we report a use case of intensified sewage surveillance to initiate public health action to thwart a looming Delta variant outbreak in Hong Kong. On 21 June 2021, albeit under basically contained COVID-19 situation in Hong Kong, routine sewage surveillance identified a high viral load of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a sewage sample from one site covering over 33,000 population, suggesting infected cases living in the respective sewershed. The use of a newly developed method based on allele-specific real-time quantitative polymerase chain reaction (AS RT-qPCR) served to alert the first documentation of the Delta variant in local community sewage three days before the case was confirmed to be a Delta variant carrier. Intensified sewage surveillance was triggered. Targeted upstream sampling at sub-sewershed areas pinpointed the source of positive viral signal across spatial scales from sewershed to building level, and assisted in determining the specific area for issuing a compulsory testing order for individuals on 23 June 2021. A person who lived in a building with the positive result of sewage testing was confirmed to be infected with COVID-19 on 24 June 2021. Viral genome sequences determined from the sewage sample were compared to those from the clinic specimens of the matched patient, and confirmed that the person was the source of the positive SARS-CoV-2 signal in the sewage sample. This study could help build confidences for public health agencies in using the sewage surveillance in their own communities.

Multiplex Epstein-Barr Virus Genotyping PCR Detects High-Risk Variants in Plasma for Population-Level Nasopharyngeal Carcinoma Screening

<h3>Purpose/Objective(s)</h3> Epstein-Barr Virus (EBV)-associated nasopharyngeal carcinoma (NPC) is unusually geographically and ethnically restricted. Without screening, most patients present at an advanced stage. Population-level screening in high-risk populations can detect most cases at an early stage, but existing serologic or molecular diagnostics are limited by low positive predictive value secondary to benign EBV reactivation. In contrast to human genome-wide association studies, recent EBV genome-wide association studies have identified polymorphisms with much greater attributable risk for NPC. We hypothesized that a noninvasive molecular diagnostic could detect high-risk EBV haplotypes in plasma to triage patients who screen positive for plasma EBV DNA. <h3>Materials/Methods</h3> We designed a multiplex allele-specific real-time polymerase chain reaction (qPCR) genotyping assay to detect three non-synonymous polymorphisms within the EBV BALF2 gene (162215C > A, 162476T > C, 163364C > T). Two dsDNA gene fragments served as either the high-risk or low-risk variant controls. Supernatant from the EBV-infected B95-8 cell line served as an additional wild-type whole-virus control. Positive clinical specimens sent for routine plasma EBV EBNA-1 testing were collected from patients with a variety of benign and neoplastic EBV-associated disorders. Three sets of conserved primers flanked the polymorphisms, with one allele-specific hydrolysis probe for each of the three high-risk alleles. A fourth allele-specific probe (162215-mt) served as an internal control. The 95% lower limit of detection (LLOD) was assessed in replicates of 20; linearity was assessed from 0.0-6.0 log10 cp/µL. Mutant allele frequencies from 0-100% were assessed by mixing dsDNA controls. <h3>Results</h3> The assay's limit of quantitation and 95% LLOD were 50 and 5 copies per well, respectively. At 100 copies per well, the assay could detect mutant allele frequencies as low as 5%, below the intra-host heterozygosity threshold. Wild-type B95-8 DNA confirmed assay specificity. Forty EBV-positive plasma specimens were genotyped from patients with non-transplant-associated viremia, post-transplant lymphoproliferative disorders, EBV-associated lymphomas (diffuse large-cell, Hodgkin, NK/T-cell, peripheral T-cell), and EBV-positive NPC. High-risk haplotypes were more common among patients with NPC (64% vs. 10%, <i>P</i> = 0.002). Compared to screening with plasma BamHI-W PCR alone, triaging screen-detected EBV DNA with this genotyping assay modestly reduced screening sensitivity (97% to 91%) but could decrease false positives and unnecessary nasoendoscopies by 63% in a modeled high-risk population. <h3>Conclusion</h3> Triaging screen-detected plasma EBV DNA with a multiplex BALF2 genotyping assay has the potential to decrease false positives and resource utilization in high-risk populations screened for NPC. This may be a low-cost and accessible alternative to higher-complexity sequencing assays in low- and middle-income countries.

The reduction of two BRCA1 gene mutations frequencies in ovarian cancer patients from Ukraine

BackgroundIn Ukraine, 3539 patients were diagnosed with ovarian cancer in 2018, and more than half of these cases were lethal. One of the risk factors for ovarian cancer development are the alterations in the breast cancer gene 1 (BRCA1). Among these alterations, the most frequent is the germline mutation 185delAG and the frameshift mutation 4153delA. The frequencies of these mutations were identified earlier (Tsip et al., 2019) in ovarian cancer patients from Ukraine. ObjectiveThe present study aimed to evaluate the frequency of the mutations 185delAG and 4153delA in the BRCA1 gene in an unselected ovarian cancer patient's cohort from Ukraine. Materials and methodsWe screened 663 ovarian cancer patients diagnosed at different ages by routine allele-specific polymerase chain reaction (PCR) and real-time PCR. Results13 cases of two BRCA1 mutations were detected (4 cases of 185delAG and 9 cases of 4153delA). ConclusionsOut of 663 ovarian cancer patients, 4 cases with 185delAG and 9 cases of 4153delA in the BRCA1 gene were identified, giving a frequency of 2.0 ± 0.5%. Our data shows that the previous mutations screening results in ovarian cancer patients from Ukraine were higher than in the present study.

Relating Interferon Regulatory Factor 5 Rs2004640 Gene Polymorphism To Increased Risk Of Systemic Sclerosis In The Patients: Russian Federation Cohort

Background ― A number of studies confirmed a crucial role of type 1 interferon in pathophysiology of connective tissue diseases. Interferon regulatory factors (IRF) coordinate an expression of type 1 interferon, while interferon regulatory factor 5 (IRF5) gene was recently identified as causing predisposition to systemic lupus erythematosus and Sjögren syndrome. The objective of our study was to identify possible association of IRF5 rs2004640 (G/T) single nucleotide polymorphism with systemic sclerosis (SSc). Material and Methods―The study involved 236 individuals, including 105 patients with SSc diagnosis and 131 control individuals from Moscow region. The latter were healthy, unrelated to each other, their genders and ages were matched to those of SSc patients. Allele-specific real-time polymerase chain reaction (PCR) was used to study IFR5 rs2004640 polymorphism. Results ― We detected significantly higher percentage of IRF5 T-allele carriers in all patients (59.5%), those with diffuse cutaneous SSc (67.3%), patients with interstitial lung lesions (62.3%), and those with positive titers of anti-topoisomerase I antibodies (66.3%), compared with control group (46.2%). The odds ratios (OR) were: 1.71 (р=0.00), 2.40 (р=0.0004), 1.93 (р=0.002), and 2.30 (р=0.0008), correspondingly. Conclusion ― The replacement of nucleotide G by T in the IRF5 rs2004640 gene polymorphism was associated with a predisposition to SSc. Our data implied an existence of a novel SSc pathogenetic pathway associated with important role of type 1 interferon in pathophysiology of connective tissue diseases.

Comparative analysis of polymorphic variants of IL-17A and MMP-1 genes with the risk of developing chronic periodontitis in petrochemical workers

The susceptibility to the development and progression of inflammatory periodontal diseases, which depends on genetic and external factors (smoking, stress, oral hygiene), varies widely. In the development of these diseases, an important role is played not only by the presence of periodontal pathogenic microorganisms, but also by the presence of congenital or acquired immunodeficiency, immunoregulatory defects. The immune system plays a key role in the physiological and pathological processes of periodontal tissues. In this regard, IL17, produced by CD4+ Th cells, which has both Pro-inflammatory and protective activity, is of particular interest in the pathogenesis of periodontitis. The aim of study was to identify the relationship between polymorphic loci of the IL-17A (rs2275913) and MMP-1 (rs1799750) genes and clinical manifestations of chronic periodontitis in petrochemical workers. Dental examination was performed in 92 ethylene oxide production workers with chronic periodontitis and 74 patients with chronic periodontitis who did not come into contact with chemical factors (control group). Genotyping of polymorphisms rs2275913 of the IL17A gene and rs1799750 of the MMP1 gene was performed by allele-specific real-time polymerase chain reaction (PCR). Hygienic assessment of the degree of air pollution of the working area with harmful substances was carried out by gas chromatography according to the guidelines for the determination of harmful substances in the air № 5098-89, № 3119-84. When comparing the results of studies of both groups, there were no statistically significant differences in the frequency distributions of allelic variants and genotypes of the IL-17A and MMP-1 genes. The AA/AG genotypes of the IL-17A gene were associated with an increased risk of severe disease compared to the GG genotype in workers in the main group (OR=6.1; 95% CI 1.33-28.5; p=0.021) and in the control group (OR=7.26; 95% CI 1.34-39.25; p=0.016). Carriers of the A allele in the control group increased the risk of severe chronic periodontitis by 2.4 times compared to carriers of the G allele (OR=2.41; 95% CI 1.19-4.87; p=0.014). During the dental examination of employees of the ethylene oxide plant, the clinical course of periodontal diseases was more severe in comparison with the control group, and the number of patients with severe periodontitis was twice as high. It was found that the AA/AG genotypes of the IL-17A gene and the carrier of the A allele are associated with increased susceptibility to the development of severe chronic periodontitis. The association between the MMP-1 gene polymorphism and the risk of severe forms of chronic periodontitis has not been established. A risk factor for the development of inflammatory periodontal diseases in employees of the petrochemical complex is a complex of harmful production factors.

Allele-Specific PCR for KRAS Mutation Detection Using Phosphoryl Guanidine Modified Primers.

Establishing the Kirsten rat sarcoma (KRAS) mutational status is essential in terms of managing patients with various types of cancer. Allele-specific real-time polymerase chain reaction (AS-PCR) is a widely used method for somatic mutations detection. To improve the limited sensitivity and specificity, several blocking methods have been introduced in AS-PCR to block the amplification of wild-type templates. Herein, we used a novel modified oligonucleotide with internucleotide phosphates reshaped 1,3-dimethyl-2-imino-imidazolidine moieties (phosphoryl guanidine (PG) groups) as primers and blockers in the AS-PCR method. Four common KRAS mutations were chosen as a model to demonstrate the advantages of the PG primers and blockers utilizing a customized PCR protocol. The methods were evaluated on plasmid model systems providing a KRAS mutation detection limit of 20 copies of mutant DNA in a proportion as low as 0.1% of the total DNA, with excellent specificity. PG-modification can serve as the universal additional mismatch-like disturbance to increase the discrimination between wild-type and mutated DNA. Moreover, PG can serve to increase primer specificity by a synergetic effect with additional mismatch and would greatly facilitate medical research.

Association of the ABCB1 C3435T gene polymorphism (SNPs) with the response to neoadjuvant chemotherapy in women with breast cancer in northeastern Brazil

&lt;p&gt;&lt;strong&gt;Introduction&lt;/strong&gt;: breast cancer (BC) is the most common tumor and the leading cause of cancer-related death among the female population&lt;br /&gt;worldwide. Polymorphisms genetics of ABCB1 gene contributed to breast cancer susceptibility and interindividual differences in&lt;br /&gt;chemotherapy response. &lt;strong&gt;Objectives&lt;/strong&gt;: to evaluate the association between the ABCB1 C3435T gene polymorphism (SNPs) with the&lt;br /&gt;response to neoadjuvant chemotherapy in women with breast cancer. &lt;strong&gt;Methodology&lt;/strong&gt;: this study included 32 female patients who&lt;br /&gt;received neoadjuvant chemotherapy. The polymorphisms were genotyped through real-time allele-specific polymerase chain reaction&lt;br /&gt;(PCR). The statistical analysis was performed using the Fisher’s exact test or Pearson’s chi-square test in the Statistical Package for&lt;br /&gt;Social Sciences (SPSS) version 20.0 software. &lt;strong&gt;Results&lt;/strong&gt;: the genotypes found for the C3435T polymorphism were in Hardy-Weinberg&lt;br /&gt;equilibrium and their genotypic distributions were CC= 10 (31.1%), CT= 14 (43.8%), and TT= 08 (25.0%) with χ2: 0.86 and p-value &amp;gt;&lt;br /&gt;0.05. Allele frequencies were C = 0.54 and T = 0.46. There were no significant statistical differences between genotypes considering the&lt;br /&gt;response to neoadjuvant chemotherapy and immunohistochemistry; the presence of the T allele was associated with worsen axillary&lt;br /&gt;status response to neoadjuvant chemotherapy. &lt;strong&gt;Conclusion&lt;/strong&gt;: no definite association between the presence of C3435T polymorphism&lt;br /&gt;and the response to neoadjuvant chemotherapy was observed. Further studies in Brazil involving larger samples will contribute to&lt;br /&gt;validating the results of this study.&lt;/p&gt;

Analysis of BRCA1 and BRCA2 genes mutations in breast cancer patients in an experiment

Aim. To assess the presence of mutations based on the analysis of the prevalence of polymorphisms in the BRCA1 and BRCA2 genes in patients admitted to the Republican clinical Oncology dispensary of the Ministry of Health of the Republic of Bashkortostan with breast cancer.&#x0D; Methods. 137 patients with breast cancer aged 25 to 80 years underwent molecular genetic testing to detect BRCA1 and BRCA2 mutations by using allele-specific real-time polymerase chain reaction. Venous blood from 105 healthy donors was used as a control group.&#x0D; Results. The study of genetic modifiers of cancer risk for BRCA1 and BRCA2 mutation carriers revealed mutations in the genes BRCA1 185delAG, 4153delA, 5382insC, T300G and BRCA2-6174 del T, which help identify genetic susceptibility to breast cancer (NSCLC). The most common form of genetic variation in patients with breast cancer was 5382insC mutation in BRCA1 gene, which was 14.59% of the total number of examined patients and 90% of the total number of positive results. BRCA1 and BRCA2 genes mutations lead to producing truncated protein, which cannot properly perform its functions and ensure DNA cell stability.&#x0D; Conclusion. Considering high breast cancer risk in BRCA1 and BRCA2 genes mutations carriers, our results show the advisability of including screening for 5382insC, 4153delA and T300G mutations in the BRCA1 gene to the screening programs for determining the risk of breast cancer.

Quantitative analysis of the BRAF V595E mutation in plasma cell-free DNA from dogs with urothelial carcinoma.

Circulating tumor DNA (ctDNA), which carries tumor-specific mutations, is an emerging candidate biomarker for malignancies and for monitoring disease status in various human tumors. Recently, BRAF V595E mutation has been reported in 80% of dogs with urothelial carcinoma. This study investigates the BRAF V595E allele concentration in circulating cell-free DNA (cfDNA) and assesses the clinical significance of BRAF-mutated ctDNA levels in canines with urothelial carcinoma. A total of 15 dogs with urothelial carcinoma were included. cfDNA concentration was measured using a real-time polymerase chain reaction (PCR) of the LINE-1 gene. To measure the concentration of the mutated BRAF gene in cfDNA, allele-specific real-time PCR with a locked nucleic acid probe was performed. BRAF mutations were detected in 11 (73%) of the 15 tested tumor samples. BRAF-mutated ctDNA concentrations were significantly higher in dogs with the BRAF mutation (14.05 ± 13.51 ng/ml) than in wild-type dogs (0.21 ± 0.41 ng/ml) (p = 0.031). The amount of BRAF-mutated ctDNA in plasma increased with disease progression and responded to treatment. Our results show that BRAF-mutated ctDNA can be detected using allele-specific real-time PCR in plasma samples of canines with urothelial carcinoma with the BRAF V595E mutation. This ctDNA analysis may be a potentially useful tool for monitoring the progression of urothelial carcinoma and its response to treatment.