Allele-specific Oligonucleotide Research Articles

Standardisation of minimal residual disease in multiple myeloma.

The assessment of the effectiveness of chemotherapy in oncology cannot disregard the concept of minimal residual disease (MRD). In fact, the efforts of numerous scientific groups all over the world are currently focusing on this issue, with the sole purpose of defining sensitive, effective assessment criteria that are, above all, able to give acceptable, easily repeatable results worldwide. Regarding this issue, especially with the advent of new drugs, multiple myeloma is one of the haematologic malignancies for which a consensus has not yet been reached. In this review, we analyse various techniques that have been used to improve the sensitivity of response, aimed at reducing the cut-off values previously allowed, as well as serological values like serum-free light chain, or immunophenotypic tools on bone marrow or peripheral blood, like multi-parameter flow cytometry, or molecular ones such as allele-specific oligonucleotide (ASO)-qPCR and next-generation/high-throughput sequencing technologies (NGS). Moreover, our discussion makes a brief reference to promising techniques, such as mass spectrometry for identifying Ig light chain (LC) in peripheral blood, and the assessment of gene expression profile not only in defining prognostic risk at the diagnosis but also as a tool for evaluation of response.

Mutation analysis of the PNPLA1 gene in a family with autosomal recessive congenital ichthyosis

Objective To identify a causative gene of autosomal recessive congenital ichthyosis (ARCI) in a Chinese family, and to analyze the genotype-phenotype correlation. Methods Peripheral blood samples were collected from the proband, his elder brother and parents, and genomic DNA was extracted from these blood samples. Genome-wide exome sequencing was conducted to determine the mutation site in the proband, and then allele-specific oligonucleotide primers were designed based on the mutation site. PCR was performed to detect the mutation site to further identify the causative gene of ARCI in the family. Results A new homozygous missense mutation was identified in exon 4 in 1 allele of the PNPLA1 gene in the proband, which led to a codon change from cytosine (C) to thymine (T) at position 700 (c.700C > T) and resulted in the substitution of proline by serine (p.pro234ser) . The same mutation was also detected in the proband′s brother, and his parents were the mutation carriers. No mutations were found in unrelated healthy Chinese individuals. Conclusion The missense mutation in the PNPLA1 gene (p.pro234ser) is associated with clinical symptoms of the patient with ARCI. Key words: Genetic diseases, inborn; Chromosome disorders; Mutation; Genes, PNPLA1; Autosomal recessive congenital ichthyosis

Cytogenetic and molecular genetic studies of number of chronic mylogenous leukemia in Erbil province

Chronic myeloid leukemia (CML) is amyeloprolirative disorder characterized by the Philadelphia chromosome originates from the translocation of chromosome 9 and 22 t (9;22) (q34;q11), creating a BCR-ABL fusion gene which results in the constitutive activation BCR-ABL tyrosine kinase. Patients of BCR-ABL fusion gene positive in (CML) are treated by an effective therapy called Imatinib (Gleevec). Point mutations alter the conformation of the ATP binding site that disturbs the binding of therapy to its target which leading to imatinib resistance. Fifty CML patients were collected from Nanakali Hospital in Erbil city which were diagnosed by physicians. The age of the patients from 9 to 80 years, 62% were male and 39% were female, median age of the patients were 38 years. The present study deals with two different aspects; conventional cytogenetic (G-banding) analysis to diagnose the Philadelphia chromosome as a (CML) marker and using Allele Specific Oligonucleotide Polymerase Chain Reaction (ASO-PCR) assay for screening the mutant allele at the three codons 351, 311 and 315 of the BCR-ABL ATP binding domain in Imatinib resistant CML patients. The phenotype of fifteen CML patients were successfully analyzed, thirteen patients were in complete cytogenetic response state (Philadelphia 0%) only two of those patients showed the Philadelphia chromosome. Three point mutations T1052C, T932C and C944T were identified by allele specific oligonucleotide polymerase chain reaction (ASO-PCR). One patient showed T1052C mutation, T932C was detected in three patients and two patients showed the C944T ATP binding domain mutation. In conclusion, conventional cytogenetic and molecular genetic tests are complementary techniques to show the exact types of abnormalities, which allow better evaluation of the genomic aberration involved in CML patients, specifically for kinase domain mutation that plays an important role in different diagnosis, prognosis, therapy treatment and drug resistant management of chronic myeloid leukemia.

A reverse dot blot assay for the screening of twenty mutations in four genes associated with NSHL in a Chinese population.

BackgroundCongenital deafness is one of the most distressing disorders affecting humanity and exhibits a high incidence worldwide. Most cases of congenital deafness in the Chinese population are caused by defects in a limited number of genes. A convenient and reliable method for detecting common deafness-related gene mutations in the Chinese population is required.MethodsWe developed a PCR-reverse dot blot (RDB) assay for screening 20 hotspot mutations of GJB2, GJB3, SLC26A4, and MT-RNR1, which are common non-syndromic hearing loss (NSHL)–associated genes in the Chinese population. The PCR-RDB assay consists of multiplex PCR amplifications of 10 fragments in the target sequence of the four above-mentioned genes in wild-type and mutant genomic DNA samples followed by hybridization to a test strip containing allele-specific oligonucleotide probes. We applied our method to a set of 225 neonates with deafness gene mutations and 30 normal neonates.ResultsThe test was validated through direct sequencing in a blinded study with 100% concordance.ConclusionsThe results demonstrated that our reverse dot blot assay is a reliable and effective genetic screening method for identifying carriers and individuals with NSHL among the Chinese population.

The Association of Angiotensinogen (AGT M235T) Gene Polymorphism and Essential Hypertension in Thai Post-Menopausal Women

Objectives: This study aims to determine the relationship between angiotensinogen (AGT) M235T polymorphism and hypertension among post-menopausal Thai women. Materials and Methods: Case-control study was conducted. The study group was those who had hypertension or previously diagnosed and, the control were those who had no hypertension. Blood samples were taken for AGT M235T allelic characterization using allele specific oligonucleotides (ASO) PCR. Results: Of 255 post-menopausal women, 128 had hypertension, regarded as “hypertension group”, the other 127 without hypertension, regarded as “control group”. The presence of AGT M235T polymorphism was 76.5% for homozygous mutation (73.4% for hypertension group and 79.5% for control group), 21.2% for heterozygous mutation (25.0% for hypertension group and 17.3% for control group, respectively) and 2.4% for homozygous wild-type (1.6% for hypertension group and 3.2% for control group, respectively). Distribution of MM, MT and TT genotypes was not significantly different between both group (p=0.251). Conclusions: Interestingly, overall TT genotype was much higher than that of TM and MM in post-menopausal Thai women. AGT M235T polymorphism was not significantly associated with hypertension, though TT genotype tended to give a small risk. They may not serve as a good genetic marker for essential hypertension among Thai population.

Quantification and Sequencing of Crossover Recombinant Molecules from Arabidopsis Pollen DNA.

During meiosis, homologous chromosomes undergo recombination, which can result in formation of reciprocal crossover molecules. Crossover frequency is highly variable across the genome, typically occurring in narrow hotspots, which has a significant effect on patterns of genetic diversity. Here we describe methods to measure crossover frequency in plants at the hotspot scale (bp-kb), using allele-specific PCR amplification from genomic DNA extracted from the pollen of F1 heterozygous plants. We describe (1) titration methods that allow amplification, quantification and sequencing of single crossover molecules, (2) quantitative PCR methods to more rapidly measure crossover frequency, and (3) application of high-throughput sequencing for study of crossover distributions within hotspots. We provide detailed descriptions of key steps including pollen DNA extraction, prior identification of hotspot locations, allele-specific oligonucleotide design, and sequence analysis approaches. Together, these methods allow the rate and recombination topology of plant hotspots to be robustly measured and compared between varied genetic backgrounds and environmental conditions.

Interleukin-8 251- A/T polymorphism related to peptic ulcer disease in H. pylori infected patient

Objectives This study includes the investigation of antifungal activity of the local propolis against dermatophytes and yeast. Methods A total of 92 tissue samples were taken from patients infected with H. pylori and 102 from uninfected individuals has been included. Genetic method used for the detection of H. pylori infection by polymerase chain reaction (PCR) for glM gene identification. IL-8- 251 A/T polymorphism was detected by allele-specific oligonucleotide polymerase chain reaction (ASO- PCR). Results Among the studied samples, the results improve that genetic polymorphism of IL-8-251 A/T genotype was found in a higher frequency with confidence interval 95% in duodenal ulcer H. pylori infected patient than control. Conclusion IL-8-251 A allele notice that it has been associated with severe inflammation lead to increase severity of disease.

Screening for a Single JAK2(p.V617F)Mutation Allele in Suspected Patients with Myeloproliferative Neoplasms (MPN) at HIWA Hospital

Detection and diagnosis of the myeloproliferative neoplasms (MPNs) are difficult to predict without screening for genetic causes, including polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF) and chronic myeloid leukemia (CML). A novel mutation in the Janus kinase 2 (JAK2) gene has been described as a genetic marker prime for all four-types of MPNs. The specific c.1851G>T (p.V617F) mutation leads to constitutively activetyrosine kinase activity of JAK2, inducing downstream JAK/STAT pathways of cytokine signaling. The existence of the JAK2(c.1851G>T; p.V617F) mutation has a clinical importance in diagnosis of MPNs. In this study, amplification refractory mutation system (ARMS) assay for a specific mutation detection was utilized, which is an Allele Specific Oligonucleotide (ASO) based multiplex PCR method. Genomic DNA samples were isolated from total blood samples from suspected MPN patients, who visited Hiwa hospital (the main teaching hospital for cancer in Sulaimani city). The aim of this study was to screen for JAK2 mutation c.1851G>T (p.V617F) on a group of suspected patients for MPNs (100 patients). The result shows that 65% of patients had indeed c.1851G>T mutation and the rest 35% of patients were normal for the mutation. In this study, we have shown the ARMS assay method to be quick, simple, cheap, reliable, and gives sufficient sensitivity for positive detection compatible for clinical diagnostic purposes. Therefore, the assay can be used for early diagnosis of MPNsin diagnostic laboratories with limited resources, such as in our health care system in our locality.

Functional GSTA1 Haplotypes Affect Clearance and Toxicity of Busulfan When Administered in 16 Doses to Pediatric Patients Undergoing Hematopoietic Stem Cell Transplantation: A Multicenter Prospective Study on Behalf of the Pediatric Disease Working Party of the European Society for Blood and Marrow Transplantation (EBMT)

Busulfan (BU) dose adjustment following therapeutic drug monitoring (TDM) contributes to better outcome of hematopoietic stem cell transplantation (HSCT). The therapeutic target window can be reached in approximately 51% to 74% of pediatric cases and 45% to 64% of infants depending on the dosing guideline used. We hypothesize that a higher proportion of pediatric patients could achieve the target BU area-under-the-curve (AUC) of 900-1500 µM.min per dose in a four times dosing schedule through genotype-guided initial BU dosing. Previous investigations by our group and others showed association of glutathione -S- transferase alpha 1 (GSTA1) with reduced BU clearance (CL), likely due to altered expression of GSTA1 enzyme, principally involved in BU metabolism.To delineate and investigate the association of each GSTA1 haplotype with the CL of BU, promoter haplotypes of GSTA1 gene were assessed in vitro with reporter gene-assay and clinically in a pediatric multi-center study (N =138) through association with BU CL, AUC, dose change and clinical outcomes. All patients received BU based myeloablative conditioning regimen. BU PK parameters from 1st dose were estimated and further doses were adjusted to have a steady state concentrations of 600-900 ng/mL and GSTA1promoter SNPs at -69, -513, -631, -1142 positions were genotyped using allele specific oligo hybridization method.Reporter-gene assays confirmed significant differences between the haplotype subgroups and supported their importance in capturing PK variability. Four GSTA1 diplotype groups (I-IV) that significantly correlated with CL (p=0.009) were distinguished. GSTA1 diplotypes (group I) underlying fast metabolizing capacity (*A2*A2&*A2*A3) had lower cumulative AUC and a higher dose increase after TDM whereas those with slow metabolizing capacity (group IV, *B1a*B1a /*B1a*B1b /*B2*B1a / *A1*B1b)had highest AUC and least dose change (p=0.02). Multivariate linear model included GSTA1 diplotype groups and non-genetic factors such as age, gender, first BU dose in mg/kg and patients origin. Dose (p<0.0005), gender (p=0.05) and GSTA1 diplotypes (p=0.01) were retained in the final model that explained 34% of overall variability. Analysis with clinical outcomes indicated higher incidences of sinusoidal obstruction syndrome in Group IV diplotype carriers (HR=7.1; 95% CI: 2.5-20.4) compared to patients with other GSTA1diplotypes. A similar association was also seen with acute graft versus host disease (aGvHD grade I-4) and combined treatment-related toxicity (TRT) (p≤0.003).In conclusion, this study has replicated and provided additional evidence for the association of GSTA1 with BU PK and clinical outcomes of HSCT. GSTA1 diplotypes can explain in some models ~20% of the variability seen in BU CL and can contribute to HSCT-related complications acting within and beyond BU metabolism. Genotyping therefore may be helpful in deciding the first BU dose, thus decreasing consequences like TRT due to inaccurate first and subsequent dosing. This may be particularly important for GSTA1 diplotype Group IV carriers. DisclosuresBittencourt:Jazz Pharmaceuticals: Consultancy, Other: Educational Grant; Seattle Genetics: Consultancy.

Triple Negative Myeloproliferative Neoplasms (MPNs) Patients Show Low MPN10 Score and Lower Grades of Bone Marrow Fibrosis

Introduction: The vast majority ofmyeloproliferative neoplasms (MPNs) patients are characterized by a molecular genetic background and by variable symptoms reflecting disease burden that may correlate with prognosis.Aim: To study the impact of triple negative status of driver gene mutations: Janus kinase 2 (JAK2), calreticulin (CALR) and myeloproliferative leukemia virus oncogene (MPL) on disease burden and its correlation with symptom severity (MPN10 score) and degree of bone marrow (BM) fibrosis in MPNs patients.Patients and Methods: MPN Symptom Assessment Form Total Symptom Score (MPN-SAF TSS) was assessed as median of 10 items: fatigue, concentration, early satiety, inactivity, night sweats, itching, bone pains, abdominal discomfort, weight loss and fever. JAK2V617F and MPL W515exon 10 mutations were performed by allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) while CALR exon 9 insertion/deletion was detected by high-resolution melting (HRM) curve analysis.Results: 100 MPNs patients (54 males and 46 females): 18 polycythemia vera (PV), 41 essential thrombocythemia (ET), 24 primary myelofibrosis (PMF), 10 Post-ET/PV-myelofibrosis (post-ET/PV-MF) and 7 myelodysplastic/myeloproliferative neoplasms (MDS/MPN) were included. Median age at diagnosis was 55 years (17-75) and was lower in ET than PV and PMF patients; 44 (19-75) years vs. 56 (34-70) years and 56 (20-75) years, respectively (p=0.06). JAK2 mutation was positive in 15 (85%) PV patients, 14 (34%) ET patients, 15 (62%) PMF patients, 8(80%) post-ET/PV-MF patients and zero (0%) MDS/MPN patients (p<0.001). CALR mutation was positive in zero (0%) PV patients, 10 (24%) ET patients, 4 (17%) PMF patients, zero (0%) Post-ET/PV-MF patients and zero (0%) MDS/MPN patients (p<0.05). MPL mutation was positive in zero (0%) PV patients, 2 (5%) ET patients, 1(4%) PMF patients, zero (0%) Post ET/PV-MF patients and zero (0%) MDS/MPN patients. Twenty four/93 (26%) patients were triple negative; 15 ET (16%), 3 PV (3%), 6 PMF (6%). Median MPN10 score was 21 (4-45) in ET versus 37.5 (25-56) in PV, 54 (15-80) in PMF and 59 (45-75) in Post-ET/PV-MF (p<0.001). Median MPN10 score was 25 (10-50) in triple negative patients vs. 40 (4-80) in MPNs patients showing at least one driver mutation positivity (p<0.001). BM fibrosis was present in 6 (15%) patients with triple negative vs. 33 (85%) patients showing at least one molecular marker positivity (p=0.007). Out of 52 patients having splenomegaly; seven (13.5%) patients were triple negative vs. 45 (87%) patients with at least one gene mutation (p<0.001). Out of the 24 triple negative patients, 19 (80%), 4 (16%), 1 (3%) and 0(0%) had BM fibrosis grades 0, 1, 2 and 3 vs. 36 (52%), 7 (10%), 12 (17%), 14 (20%) out of 69 patients with at least one gene mutation, respectively (p=0.002). After a median follow-up period of 16 months (3-151), overall survival (OS) was 95%. OS in PV and ET patients was 100 % versus 83 % in PMF patients (p=0.08). OS in triple negative group was 100% versus 94% in the gene mutations group (p=0.387).Conclusion: Driver gene mutations show an impact on disease symptoms and burden. Triple negative MPNs patients in our cohort have significantly low MPN10 score and less BM fibrosis which may indicate a more indolent disease course. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Development of Novel Human Anti-HLA-Monoclonal Antibodies for Clinical Applications Using Peripheral Blood B Cells Derived from Anti-HLA Antibody-Positive Donors

Introduction: The development of monoclonal antibodies (mAbs) such as anti-CD20 mAb (rituximab) and anti-CD38 mAb (daratumumab) has revolutionized the treatment of lymphoid malignancies. Potential efficacy of HLA allele-specific mAbs in treating malignant lymphoma has been shown by several studies. However, treatment of B-cell malignancies with humanized mAbs against HLA-DR alleles is associated with infusion-related toxicities (Lin et al., Leuk Lymphoma, 2009). In addition, previous attempts to develop mAbs specific to some HLA alleles (e.g., HLA-B61) by immunizing mice with recombinant HLA proteins have failed, likely because of a wide variety of polymorphisms of HLA molecules. To overcome these problems, we recently created a novel method to develop mAbs using microarray technology and human peripheral blood (PB) B lymphocytes derived from donors who are positive for anti-HLA antibodies. The process took approximately 1 month to produce human anti-HLA mAbs.Methods: Approximately 20 ml of PB was collected from anti-HLA antibody-positive donors, and mononuclear cells (MNCs) isolated using the Ficoll-Hypaque method were cultured in RPMI-1640 + 10% FCS containing 5 μg/ml R-848, 1000 IU/ml hIL2, 2.5 μg/ml CpG2006, 2.5 μg/ml anti-CD40 antibody, 100 ng/ml hIL21, 2 ng/ml hIL17, 10 ng/ml hIL4, and 100 ng/ml hBAFF for 6 days. CD138+ cells were then isolated using anti-CD138-antibody-conjugated microbeads. A microwell array chip was manufactured using micromachining techniques, as previously described (Jin et al., Nature Medicine, 2009). Microwells (diameter, 10 μm; depth, 15 μm) were formed on a silicon surface. The chip was coated with a PBS-containing purified HLA antigen (10 μg/ml) and incubated overnight at 4°C. After removing the antigen solution, 50 μl of the CD138+ cell suspension was added to the chip, and the mixture was incubated for 4 h at 37°C. After gentle washing with PBS, 2 μg/ml of anti-human IgG Fc-Cy3 solution was added to the microwells, and the plate was left at room temperature for 15 min. Finally, the cells were stained with 1 μM Oregon Green for 2 min. The microwells were screened for the antigen-specific antibodies released from single cells under a fluorescence microscope. Antigen-specific antibody-secreting cells (ASCs) from individual wells were isolated using a micromanipulator fitted with capillaries under the fluorescence microscope and were then expelled to microtubes for reverse transcription. The antibody cDNA fragments for VH and VL fragments were amplified using the single-cell 5′-RACE method and inserted into expression vectors containing the complete constant region of cDNA for heavy or light chains. Thereafter, CHO cells were transfected with both the heavy and light chain expression vectors to obtain a supernatant containing complete antibody molecules. The antigen specificity of the recombinant antibodies was examined using ELISA and flow cytometry (FCM). The frequency of ASCs in the PB-MNCs of donors was quantified using allele-specific oligonucleotide PCR. Complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) of the mAbs were assessed using conventional methods.Results and Discussion: Two novel human mAbs specific to HLA-A24 and HLA-B61 were successfully generated. The antigen specificity of the mAbs was confirmed using ELISA and FCM. The mAbs bound to normal and malignant blood cells that expressed corresponding HLA alleles and showed CDC/ADCC activities against five B lymphoblastoid cell lines but not against lymphocytes/monocytes/granulocytes derived from five healthy donors, probably because of low expression levels of the target HLA alleles. The frequency of ASCs in PB-MNCs of donors was less than 0.001%.Conclusions: The present method enabled the generation of mAbs specific to HLA alleles, which can be used for detecting minimal residual diseases of hematological malignancies as well as for treating B-cell lymphoma, within 1 month. DisclosuresTakamatsu:Celgene: Honoraria; Janssen Pharmaceuticals: Honoraria. Kawai:Wakunaga Pharmaceutical Co., Ltd.: Employment. Nakao:Alexion Pharmaceuticals: Honoraria, Research Funding.

Silencing of microRNA-138-5p promotes IL-1β-induced cartilage degradation in human chondrocytes by targeting FOXC1: miR-138 promotes cartilage degradation.

ObjectivesOsteoarthritis (OA) is characterised by articular cartilage degradation. MicroRNAs (miRNAs) have been identified in the development of OA. The purpose of our study was to explore the functional role and underlying mechanism of miR-138-5p in interleukin-1 beta (IL-1β)-induced extracellular matrix (ECM) degradation of OA cartilage.Materials and MethodsHuman articular cartilage was obtained from patients with and without OA, and chondrocytes were isolated and stimulated by IL-1β. The expression levels of miR-138-5p in cartilage and chondrocytes were both determined. After transfection with miR-138-5p mimics, allele-specific oligonucleotide (ASO)-miR-138-5p, or their negative controls, the messenger RNA (mRNA) levels of aggrecan (ACAN), collagen type II and alpha 1 (COL2A1), the protein levels of glycosaminoglycans (GAGs), and both the mRNA and protein levels of matrix metalloproteinase (MMP)-13 were evaluated. Luciferase reporter assay, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot were performed to explore whether Forkhead Box C1 (FOCX1) was a target of miR-138-5p. Further, we co-transfected OA chondrocytes with miR-138-5p mimics and pcDNA3.1 (+)-FOXC1 and then stimulated with IL-1β to determine whether miR-138-5p-mediated IL-1β-induced cartilage matrix degradation resulted from targeting FOXC1.ResultsMiR-138-5p was significantly increased in OA cartilage and in chondrocytes in response to IL-1β-stimulation. Overexpression of miR-138-5p significantly increased the IL-1β-induced downregulation of COL2A1, ACAN, and GAGs, and increased the IL-1β-induced over expression of MMP-13.We found that FOXC1 is directly regulated by miR-138-5p. Additionally, co-transfection with miR-138-5p mimics and pcDNA3.1 (+)-FOXC1 resulted in higher levels of COL2A1, ACAN, and GAGs, but lower levels of MMP-13.ConclusionmiR-138-5p promotes IL-1β-induced cartilage degradation in human chondrocytes, possibly by targeting FOXC1.Cite this article: Y. Yuan, G. Q. Zhang, W. Chai,M. Ni, C. Xu, J. Y. Chen. Silencing of microRNA-138-5p promotes IL-1β-induced cartilage degradation in human chondrocytes by targeting FOXC1: miR-138 promotes cartilage degradation. Bone Joint Res 2016;5:523–530. DOI: 10.1302/2046-3758.510.BJR-2016-0074.R2.

Dasatinib and low-intensity chemotherapy in elderly patients with Philadelphia chromosome–positive ALL

AbstractPrognosis of Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) in the elderly has improved during the imatinib era. We investigated dasatinib, another potent tyrosine kinase inhibitor, in combination with low-intensity chemotherapy. Patients older than age 55 years were included in the European Working Group on Adult ALL (EWALL) study number 01 for Ph+ ALL (EWALL-PH-01 international study) and were treated with dasatinib 140 mg/day (100 mg/day over 70 years) with intrathecal chemotherapy, vincristine, and dexamethasone during induction. Patients in complete remission continued consolidation with dasatinib, sequentially with cytarabine, asparaginase, and methotrexate for 6 months. Maintenance therapy was dasatinib and vincristine/dexamethasone reinductions for 18 months followed by dasatinib until relapse or death. Seventy-one patients with a median age of 69 years were enrolled; 77% had a high comorbidity score. Complete remission rate was 96% and 65% of patients achieved a 3-log reduction in BCR-ABL1 transcript levels during consolidation. Only 7 patients underwent allogeneic hematopoietic stem cell transplantation. At 5 years, overall survival was 36% and up to 45% taking into account deaths unrelated to disease or treatment as competitors. Thirty-six patients relapsed, 24 were tested for mutation by Sanger sequencing, and 75% were T315I-positive. BCR-ABL1T315I was tested by allele-specific oligonucleotide reverse transcription–quantitative polymerase chain reaction in 43 patients and detection was associated with short-term relapses. Ten patients (23%) were positive before any therapy and 8 relapsed, all with this mutation. In conclusion, dasatinib combined with low-intensity chemotherapy was well-tolerated and gave long-term survival in 36% of elderly patients with Ph+ ALL. Monitoring of BCR-ABL1T315I from diagnosis identified patients with at high risk of early relapse and may help to personalize therapy.

Gene Polymorphisms and Serum Levels of Pro- and Anti-Inflammatory Markers in Dengue Viral Infections.

Pro- and anti-inflammatory markers (tumor necrosis factor [TNF]-α, TNF-β, interferon [IFN]-γ, interleukin [IL]-6, IL-8, IL-10, and C-reactive protein [CRP]) were investigated in 80 patients infected with dengue viruses, 100 patients presenting with febrile illness but negative for dengue, and 99 healthy subjects. Immunoenzyme methods were used for quantitative assays in the plasma. Polymorphisms of TNF-α, TNF-β, IL-6, IL-8, and IL-10 genes were assessed by polymerase chain reaction (PCR)-restriction fragment length polymorphism and allele-specific oligonucleotide (ASO)-PCR for the IFN-γ. The highest mean serum levels of TNF-α, IFN-γ, IL-8, and CRP were observed in dengue-positive individuals. TNF-β, IL-6, and IL-10 levels were significantly higher in the dengue-negative individuals. No cytokine expression pattern was evidenced according to virus serotype. Genotypic frequency distributions were statistically significant for the polymorphisms of TNF-α and IFN-γ among positive, negative, and control dengue groups and IFN-γ among groups DENV-1, DENV-2, DENV-3, and controls. Modulation of cytokine expression and polymorphisms is a complex matter and needs further explanation considering the ethnic origins of the Brazilian population.

Time of onset of coronary artery disease in diabetic patients depends on genetic polymorphism in region 9p21 but not in 1p25

Introduction. Type 2 diabetes (T2DM) is an important risk factor for development of coronary artery disease (CAD) and its complications. Recent genetic studies revealed possible association between those two conditions on the genome level. In our study we analysed whether polymorphisms in loci 1q25 and 9p21, previously characterized as risk factors of CVD, have an influence on early-onset CAD in T2DM patients. Materials and methods. Our case-control study included 338 patients suffering from T2DM and CAD. For the study purpose the cohort was divided into two groups based on the age of CAD onset: case group with earlyonset CAD (< 55 for males and < 65 years for females, n = 180) and control group (≥ 55 and ≥ 65 years respectively, n = 158). Epidemiological data was taken from medical history and retrospective questionnaire; blood samples were collected. The rs2383206, rs1333049 and rs10911021 were genotyped using method of fluorescently labelled allele specific oligonucleotides. Results. Statistical analysis did not reveal any significant differences between two groups in the mean duration of diabetes, metabolic parameters (weight, waist circum- ference, frequency of obesity according to BMI, mean blood pressure or lipids levels) and smoking history. There were statistical differences between groups in family incidence of CAD (70.0% patients in cases vs. 45.6% in controls; p < 0.005), hypertension (77.1% vs. 47.8%; p < 0.005) and obesity (61.2% vs. 49.0%; p < 0.05). Genetic analysis revealed that frequency of the G allele of rs2383206 in 9p21 region was significantly higher in cases than in controls (62.4% vs 44.0% p < 0.00001). Homozygotes GG were 39.4% of cases and 18.8% in controls (p = 0.0001). The OR for increased risk of early CAD in GG homozygotes was 2.81, 95% CI: 2.39–3.24, after adjustment for conventional risk factors it was reduced to 2.58. There was statistically significantly higher frequency of GG homozygotes among patients with poor glycaemic control (HbA1c ≥ 7%; 41.3% vs. 15.9; p = 0.0011) and non-obese subjects (BMI < 30 kg/m2; 39.7% vs. 18.2%, p = 0.0002). Similar association between genotype and risk of early CAD was found for another polymorphism rs1333049 in 9p21 region. Frequency of allele C was significantly higher in cases than in controls (56.3% vs 43.4, p = 0.0036) and homozygotes CC were 31.6% in cases and 17.4% in controls (p = 0.0083). OR for this association was 2.2 (95% CI: 1.83–2.56); after adjustments 1.96. We didn’t find any association between genotypes distribution of rs10911021 and early onset of CAD. Conclusions. Our findings clearly suggest that polymorphisms in 9p21 region have an influence on development of early-onset CAD in T2DM, especially in non-obese patients and subjects with poor glycaemic control. (Clin Diabet 2016; 5, 1: 7–14)

Identification of the Dominant Genotypes of Phytophthora infestans in Canada Using Real-Time PCR with ASO-PCR Assays.

Phytophthora infestans, a pathogenic oomycete that is the causal agent of potato and tomato late blight, has devastating effects worldwide. The genetic composition of P. infestans populations in Canada has changed considerably over the last few years, with the appearance of several new genotypes showing different mating types and sensitivity to the fungicide metalaxyl. Genetic markers allowing for a rapid assessment of genotypes from small amounts of biological material would be beneficial for the early detection and control of this pathogen throughout Canada. Mining of the P. infestans genome revealed several regions containing single-nucleotide polymorphisms (SNP) within both nuclear genes and flanking sequences of microsatellite loci. Allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) assays were developed from 14 of the 50 SNP found by sequencing. Nine optimized ASO-PCR assays were validated using a blind test comprising P. infestans and other Phytophthora spp. The assays revealed diagnostic profiles unique to each of the five dominant genotypes present in Canada. The markers developed in this study can be used with environmental samples such as infected leaves, and will contribute to the genomic toolbox available to assess the genetic diversity of P. infestans at the intraspecific level. For late blight management, early warning about P. infestans genotypes present in potato and tomato fields will help growers select the most appropriate fungicides and application strategies.

Minimal Residual Disease Assessment in the Context of Multiple Myeloma Treatment.

With contemporary therapeutic strategies in multiple myeloma, heretofore unseen depth and rate of responses are being achieved. These strategies have paralleled improvements in outcome of multiple myeloma patients. The integration of the next generation of proteasome inhibitors and antibody therapeutics promise continued improvements in therapy with the expectation of consistent depth of response not quantifiable by current clinical methods. As such, there is a growing need to develop adequate tools to evaluate deeper disease response after therapy and to refine the response criteria including the minimal residual disease. Several emerging techniques are being evaluated for these purposes including multi-parameter flow cytometry, allele-specific oligonucleotide polymerase chain reaction, next-generation sequencing, and imaging modalities. In this review, we highlight the recent developments and evaluate advantages and limitations of the current technologies to assess minimal residual disease. We also discuss future applications of these methodologies in potentially guiding multiple myeloma treatment decisions.

LGALS3 and AXIN1 gene variants playing role in the Wnt/ β-catenin signaling pathway are associated with mucinous component and tumor size in colorectal cancer

The Wnt pathway alterations have been identified in colorectal and many other cancer types. It has been reported that galectin-3 (which is encoded by the LGALS3 gene) alters the signaling mechanism in the Wnt/β-catenin pathway by binding to β-catenin in colon and other cancers. AXIN1 is mainly responsible for the assembly of the β-catenin destruction complex in the Wnt pathway. This study investigated the relationship of rs4644 and rs4652 variants of the LGALS3 gene and rs214250 variants of the AXIN1 gene to histopathological and clinical properties. Our study included a total of 236 patients, of whom 119 had colorectal cancer (42 women, 77 men) and 117 were healthy controls. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and allele-specific oligonucleotide (ASO) PCR methods were used. In addition, the serum galectin-3 level was studied with the enzyme-linked immunosorbent assay (ELISA) method. For the rs4644 variant of the LGALS3 gene, the CC genotype a mucinous component was significantly more common than those without a mucinous component (p=0.026). C allele frequency of the rs214250 variant of the AXIN1 gene was significantly correlated to tumor size in the advanced tumor stage (p=0.022). The CCAACT haplotype was more common in colorectal cancer patients (p=0.022). Serum galectin-3 level was higher in the patient group compared to the control group (5.9± 0.69 ng/ml vs. 0.79±0.01 ng/ml; p<0.001). In conclusion, variants of LGALS3 and AXIN1 genes affect tumor sizes and the mucinous component via Wnt/β-catenin pathway in the pathogenesis of colorectal cancer.

Genotyping of single nucleotide polymorphisms related to attention-deficit hyperactivity disorder.

Pharmacological treatment of several diseases, such as attention-deficit hyperactivity disorder (ADHD), presents marked variability in efficiency and its adverse effects. The genotyping of specific single nucleotide polymorphisms (SNPs) can support the prediction of responses to drugs and the genetic risk of presenting comorbidities associated with ADHD. This study presents two rapid and affordable microarray-based strategies to discriminate three clinically important SNPs in genes ADRA2A, SL6CA2, and OPRM1 (rs1800544, rs5569, and rs1799971, respectively). These approaches are allele-specific oligonucleotide hybridization (ASO) and a combination of allele-specific amplification (ASA) and solid-phase hybridization. Buccal swab and blood samples taken from ADHD patients and controls were analyzed by ASO, ASA, and a gold-reference method. The results indicated that ASA is superior in genotyping capability and analytical performance.

BRCA1 founder mutations do not contribute to increased risk of gastric cancer in the Polish population

BackgroundGastric cancer (GC) is part of the spectrum of diseases linked to BRCA1 and BRCA2 mutations that increase the risk of breast and ovarian cancer. Data suggesting an increased risk of developing GC among BRCA1 and BRCA2 mutation carriers are based almost exclusively on indirect studies. The objective was to assess in a direct study whether there is a relationship between GC and selected recurrent BRCA1 and BRCA2 mutations in the Polish population.MethodsThree hundred seventeen GC patients (193 males and 124 females; mean age 59.5 ± 12.8 y) diagnosed at the Department of Gastroenterology at the Pomeranian Medical University were included in this retrospective study. All patients were genotyped for 3 BRCA1 Polish founder mutations (5382insC, C61G and 4153delA) as well as for 9 known recurrent mutations in BRCA1 and BRCA2 genes. Genotyping was performed using allele-specific oligonucleotide polymerase chain reaction (ASA-PCR) for 4153delA and 5382insC, restriction fragment length polymorphism (PCR-RFLP) for C61G and TaqMan real-time PCR for 185delAG, 3819del5, 3875del4, 5370C > T, 886delGT, 4075delGT, 5467insT, 6174delT and 8138del5.ResultsAmong tested mutations one founder BRCA1 mutation 5382insC was detected in two of 317 (0.63 %) GC cases. A comparison of frequency of detected BRCA1 founder mutations in GC patients to previously described 4570 Polish controls (0.63 % vs. 0.48 %) failed to indicate an increased risk of GC in the mutation carriers (OR = 1.3; 95 % CI 0.3-5.6, p = 0.71). A comparison of frequency of GC male cases and male controls (1.0 % vs. 0.43 %,OR = 1.5; 95 % CI 0.3-6.4, p = 0.61) allowed to formulate the same conclusion that there is no increased risk for GC for males. None of the 9 recurrent BRCA1 and BRCA2 mutations has been detected in tested GC patients.ConclusionThe current study indicates that founder BRCA1 mutations reported in Polish breast/ovarian cancer patients do not contribute to increased GC risk. The nine tested recurrent BRCA1 and BRCA2 mutations were not detected in GC patients which may suggests that they are rare in GC patients in the Polish population. Further analyses, including sequencing of entire sequences of BRCA1 and BRCA2 genes, are necessary to ultimately determine the role of these two genes in GC in Poland.