scholarly journals PCR đặc hiệu allele tích hợp công nghệ amplification refractory mutation system (Dsa-Pcr-Arms) phát hiện đột biến jak2 v617f và Carl trên bệnh nhân có hội chứng tăng sản tủy

2020 ◽  
Author(s):  
Tất Trung Ngô
Keyword(s):  
Molecular Genetic ◽  
Myeloproliferative Neoplasm ◽  
Jak2 V617f ◽  
Positive Sample ◽  
Amplification Refractory Mutation System ◽  
Study Cohort ◽  
Specific Pcr ◽  
Mutation System ◽  
Allele Specific ◽  
Allele Specific Pcr

BIDIRECTIONAL ALLELE SPECIFIC PCR INTEGRATED WITH AMPLIFICATION REFRACTORY MUTATION SYSTEM –(DSA-PCR-ARMS) FOR ANALYZING CARL AND JAK2 V617F MUTATIONS IN PATIENTS WITH MYELOPROLIFERATIVE NEOPLASM Bachground: Jak2 V617F and CARLmutation arerecognized as essential molecular genetic markers in diagnostic definition of myeloid proliferative neoplasm (MPN). Objective: to establish an income relevant Allele-specific-PCR-ARMS (DSA-PCR-ARMS) assays to identify Jak2 V617F, CARL mutations in MPN patients. Subjects: 64 MPN patients’ samples, one V617F positive referrance samples and one CALR mutant positive sample. Methods: dilution series of 100%, 50%, 25%, 5%, 0,5% and 0% mutant alleles were created as pseudo-samples establish the detection limits and the technical specificity of DSA-PCR-ARMS assays in identifying the mentioned genetics markers. Result: Our approach can detect presence of 0.5 % V617F mutant DNA fragment; beside that just by one gel-based PCR reaction, we can differentiate the deletion/insertion CARL carriers from the wild type counterpart. By applying our approachesto a study cohort of 50 MPN cases, a sensitivity of 64% and specificity of 100% was recorded for our Jak2 V617F detection assay and we also detected 3 CARL mutation case amongst the 10 V617F negative cases. Conclusion: The in-house of Jak2 V617F/CARL mutation detection protocols have been successfully established in our hospital. Key words: Jak2 V617F, CARL, myeloid proliferative neoplasm (MPN).

Presence of JAK2 V617F Mutation in Peripheral Blood of Blood Donors with Upper-Limit Hematocrit and Platelet Values.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2533-2533
Author(s):  
Paola Bianchi ◽  
Elisa Fermo ◽  
Fulvio Mozzi ◽  
Maurizio Marconi ◽  
Alberto Zanella
Keyword(s):  
Polycythemia Vera ◽  
Blood Donors ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Myeloid Metaplasia ◽  
Specific Pcr ◽  
Upper Limit ◽  
Allele Specific ◽  
V617f Mutation ◽  
Allele Specific Pcr

Abstract The somatic mutation V617F of JAK2 gene has been identified as a pathogenic factor in typical chronic myeloproliferative diseases (MPDs), in particular polycythemia vera, essential thrombocythemia, and myelofibrosis with myeloid metaplasia. Recently, two studies showed the presence of this mutation also in 37/3935 subjects with non haematological diseases (Xu et al, 2006) and 5/52 healthy donors (Sidon et al, 2006), suggesting that V617F mutation may occur in the absence of MPD phenotype and that probably is not sufficient per se to induce MPDs. The aim of this study was to search for the presence of JAK2 V617F mutation in healthy blood donors with confirmed upper-limit Hct and/or Plts values. Actually, previous studies indicated that some subjects with upper-limit Hct levels have early stages of polycythemia vera (Zanella et al, 1987). We studied 177 consecutive repeat blood donors (92 M, 85 F; median age 45 years, range 19–66) displaying Hct and/or Plts values higher than the 75° percentile of the normal reference distribution (Hct > 0.47 for M and > 0.42 for F; Plts > 300×109/L), confirmed on at least two different occasions in the last 12 months. All subjects had been accepted for blood donation on the basis of negative clinical history and normal results on both physical examination and routine laboratory testing. 83 of them (55 M and 28 F) had upper-limit Hct levels (median 0.48, range 0.47-0.51 for M; 0.43, range 0.42-0.47 for F); 85 had Plts > 300×109/L (median 338×109/L, range 300–454), and 9 donors had both upper-limit Hct and Plts. DNA was extracted from whole blood; all samples were analyzed by allele-specific polymerase chain reaction (PCR) according to Baxter et al (2005), and by fluorescent allele specific PCR (McClure et al, 2006) on ABI PRISM 310 Genetic Analyzer. Ten subjects were found to be positive for V617F mutation by fluorescent PCR, showing a positive signal when compared to a positive control corresponding to 2% of V617F mutated allele. Six of them showed a positive band also on agarose gel when analyzed with allele specific PCR. The presence of mutation was confirmed by enzymatic digestion with BsaXI. Hematological data of mutated subject are reported in the table. No statistically significant differences of hematological parameters were present between V617F positive and negative subjects. In conclusion, the presence of a V617F positive clone (albeit in a small amount), was found in 4% (3 F and 1 M) donors with upper-limit Hct and in 6% (2 F, 4 M) donors with Plts > 300×109/L. The follow up of these subjects will ascertain whether V617F mutation is a prelude to a myeloproliferative disease. Sex Age (years) Hb (g/dl) Hct Plts (×109/L) WBC (x109/L) Upper-limit Hct 1 F 66 15.1 0.45 202 4.85 2 F 51 14.4 0.43 235 6.40 3 F 64 15.7 0.45 198 7.75 4 M 58 15.9 0.48 220 7.30 Plts > 300×109/L 5 F 53 13.7 0.40 360 6.97 6 F 63 13.5 0.40 301 9.2 7 M 47 15.2 0.45 334 8.64 8 M 47 13.8 0.41 316 6.35 9 M 19 15.2 0.44 321 8 10 M 37 16.1 0.45 379 7.9

Plasma Detection of JAK2 Mutation Is Not Suitable as a Clinical Test

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5231-5231
Author(s):  
Mohamed E Salama ◽  
Sabina Swierczek ◽  
Roberto H Nussenzveig ◽  
Kimberly Hickman ◽  
Andrew Wilson ◽  
...  
Keyword(s):  
Quantitative Assessment ◽  
Jak2 V617f ◽  
Progressive Increase ◽  
Clinical Test ◽  
Jak2 Mutation ◽  
Specific Pcr ◽  
Highly Sensitive ◽  
Allele Specific ◽  
Allele Specific Pcr ◽  
Over Time

Abstract The 1849G>T (V617F) JAK2 gene activating point mutation is identified in a substantial number of chronic myeloproliferative neoplasms (MPN) and the mutant allele burden correlates with some of the complications of MPN, justifying the introduction of quantitative JAK2 assays in clinical practice. Recent reports suggested a higher detection rate of the JAK2 mutation using plasma rather than by cell analysis and even suggested its use to detect zygosity. In this study we questioned the biologic basis of this suggestion and tested its validity by comparing JAK2 mutation allele burden in paired cell and plasma samples over time using a highly sensitive quantitative assay. Peripheral blood samples were collected from known patients with a positive JAK2 mutation as detected in their purified granulocytes. Separation of plasma and granulocytes (GNC) were carried on day 0,3,5,7,9,10 after sample collection. Quantitative assessment of JAK2 was performed on a total of 63 samples from 7 patients using allele-specific PCR amplification of JAK2-exon14 in genomic DNA isolated from the paired purified peripheral blood granulocytes and plasma samples. The detection of wild and mutant JAK2 alleles was achieved by highly sensitive and discriminatory allele-specific PCR utilizing allele specific primers that incorporate a mismatched nucleotide and synthetic ”locked nucleic acid” (Nussenzveig Expl Hemat.35: 32–38, 2007). Quantitative assessment of the sample is achieved by real-time monitoring amplification from each allele (in separate reactions). The values for each reaction are then used to determine the frequency of the T-allele relative to the G-allele in the sample and statistical calculations were performed using SAS software, Version 9.1 of the SAS System Copyright © 2002–2003 SAS Institute Inc, Cary, NC, USA. Quantitative measurements of JAK2 V617F mRNA in these samples were also analyzed. The JAK2 V617F range was (0.2–90%) in GNC and (0–88%) in plasma with no concordance of corresponding values in any patient at any given time point. We identified a progressive increase in plasma JAK2 V617F DNA values accompanied by a progressive reciprocal decrease of JAK2 V617F in paired GNC samples in all cases over time, suggesting preferential lysis of the GNC bearing the JAK2 V617F mutation. Similar data were obtained using in vitro expansion of JAK2 V617F polycythemia vera erythroid progenitors. A repeated-measures ANOVA was calculated to determine whether there was interaction between GNC vs. plasma and time. The rate of change in the value of JAK2 did indicate an interaction and was significantly different (p=.003) between GNC and plasma. In one patient, the JAK2 V617F was detected in GNC (0.7%) but not in plasma sample on day 0. We conclude that detection of JAK2 V617F in plasma is due to decreased viability of the JAK2 bearing cells, which leads to progressive increase in plasma detection. There is no correlation between JAK2 V617F allelic burden values in GNC and plasma at any given point. JAK2 quantitation in plasma is not suitable as a clinical test.

scholarly journals Rapid characterization of b-thalassemia mutations by reverse dot blot and allele-specific pcr analysis

2002 ◽  
Vol 21 (3) ◽  
pp. 283-286 ◽  
Author(s):  
Sonja Pavlovic ◽  
Jelena Urosevic ◽  
Tatjana Djureinovic ◽  
Dragana Janic ◽  
Lidija Krivokapic-Dokmanovic
Keyword(s):  
Pcr Analysis ◽  
Amplification Refractory Mutation System ◽  
Thalassaemia Major ◽  
Dot Blot ◽  
Specific Pcr ◽  
Mutation System ◽  
Allele Specific ◽  
The Republic ◽  
Rapid Characterization ◽  
Reverse Dot Blot

This paper reports a case of b-thalassaemia major whose molecular diagnosis was achieved by using modern methods of molecular genetics. This example demonstrates the strategy we chose to detect b-thalassaemia mutations in the Republic of Serbia in order to complete molecular screening in our population and to make prenatal diagnosis in pregnancies at risk. The analysis of genomic DNA isolated from the blood of patient affected with thalassaemia major is carried out by the methods: RDB (reverse dot blot) and ARMS (amplification refractory mutation system). It is shown that the patient is a compound heterozygote for two b- -thalassaemic mutations: b+IVSI-110 and b+IVSI-6.

A Novel and Quantitative Assay To Detect JAK2 V617F Allele by Real-Time AS-PCR and Its Applicability to PV Initiating Mutation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4915-4915
Author(s):  
Roberto H. Nussenzveig ◽  
Sabina Swierczek ◽  
Jaroslav Jelinek ◽  
Srdan Verstovsek ◽  
Jaroslav Prchal ◽  
...  
Keyword(s):  
Real Time ◽  
Allele Frequency ◽  
Somatic Mutation ◽  
Mutant Allele ◽  
Jak2 V617f ◽  
Allele Frequencies ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr

Abstract Polycythemia vera (PV) arises due to a somatic mutation(s) of a single hematopoietic stem cell leading to clonal hematopoiesis. Greater than 80% of PV patients carry a somatic mutation in JAK2 (V617F). Growing evidence suggests that increased frequency of the JAK2V617F allele may have a prognostic impact on certain clinical aspects of PV, and, possibly, in other myeloproliferative disorders associated with this mutation. We have developed a novel approach to primer design for Real-Time quantitative allele-specific PCR. Allelic discrimination is enhanced by the combined synergistic effects of an artificial mismatch introduced in the −1 position, starting from the 3′ end of the primer, and the use of a locked nucleic acid (LNA) modified nucleoside placed at the −2 position. We provide evidence that the −2 LNA assists in stabilizing the 3′ end, while the −1 mismatch provides specificity but not stability. The difference in cycle number between the two allele-specific reactions is used to calculate the relative allele frequencies. We demonstrate the robustness, sensitivity and reproducibility of our design. The proportion of mutant JAK2 allele determined by pyrosequencing and kinetic allele-specific PCR was highly concordant with an average allele frequency deviation of 2.6%. Repeated determination of allelic ratios in multiple patient samples was highly reproducible with a standard deviation of 1.5%. We have also determined that the design and assay is highly sensitive; as little as 0.1% mutant allele in 40–50 ng of genomic DNA can be detected. We further tested the applicability of this technique to the analysis of individual BFU-E colonies in order to address the question whether the JAK2V617F is the disease initiating mutation. Less than 10% of a single isolated BFU-E colony, originating from a single progenitor, is sufficient for determination of allele frequency. The remainder of the colony may be used for other analyses. A proportion of 0 or 50 or 100 percent JAK2 mutant allele is expected from each individual BFU-E colony, which was indeed observed. However, when we tested granulocytes from PV females, wherein the granulocytes were found to be clonal by the X-chromosome transcriptionally based clonality assay, we found 3 females <50 (27.5 ±11) and 7 females >50 (75 ±10.5)percent mutant JAK2 allele frequencies. This result suggests the presence of a heterogeneous population of cells with differing genotypes regarding the JAK2 mutant allele, and is further supported by our genotyping results with individual BFU-E colonies as described above. Our PV data suggest that the JAK2V617F may not be the PV initiating mutation. This novel primer design is simple, does not require tedious optimization of reaction conditions, and can be applied to any kinetic PCR platform for reliable and sensitive determination of allele frequencies. Potential applications are varied, such as, quantitative determination of mosaicism, proportion of fetal cells in maternal circulation, detection of minimal residual disease associated with known somatic mutation (such as reduction of malignant cells by chemotherapy or reappearance of resistant clone), rapid monitoring of efficacy of new drugs in both “in vitro” systems as well as clinical trials, and many others that require quantitation of allele frequencies.

scholarly journals Multiplexed Assays for Detection of Mutations in PIK3CA

2008 ◽  
Vol 54 (4) ◽  
pp. 757-760 ◽  
Author(s):  
Ruth E Board ◽  
Nicola J Thelwell ◽  
Paul F Ravetto ◽  
Stephen Little ◽  
Malcolm Ranson ◽  
...  
Keyword(s):  
Pik3ca Mutation ◽  
Clinical Samples ◽  
Amplification Refractory Mutation System ◽  
Pik3ca Mutations ◽  
Specific Pcr ◽  
Mutation System ◽  
Allele Specific ◽  
Phosphoinositide 3 Kinase ◽  
Low Sensitivity ◽  
Detection Of Mutations

Abstract Background: Mutations in the PIK3CA gene (phosphoinositide-3-kinase, catalytic, alpha polypeptide) have recently been described in a number of cancers, and their detection is currently limited because of the low sensitivity of conventional sequencing techniques. Methods: We combined Amplification Refractory Mutation System (ARMS™; AstraZeneca) allele-specific PCR and Scorpions™ (DxS) to develop assays for tumor-borne PIK3CA mutations and used real-time PCR to develop high-throughput multiplexed assays for the most commonly reported PIK3CA mutants (H1047L, H1047R, E542K, E545K). Results: These assays were more sensitive than sequencing and could detect 5 copies of mutant DNA in proportions as low as 0.1% of the total DNA. We assayed DNA extracted from human tumors and detected PIK3CA mutation frequencies of 10.2% in colorectal cancer, 38.7% in breast cancer, 1.9% in lung cancer, and 2.9% in melanoma. In contrast, sequencing detected only 53% of the mutations detected by our assay. Conclusions: Multiplexed assays, which can easily be applied to clinical samples, have been developed for the detection of PIK3CA mutations.

JAK2, CALR, MPL, and ASXL1 Mutations in 136 Thai Patients with Philadelphia-Negative Myeloproliferative Neoplasms and Their Correlations with Clinical Outcomes

2021 ◽  
Vol 104 (5) ◽  
pp. 834-845
Keyword(s):  
Clinical Outcomes ◽  
Myeloproliferative Neoplasms ◽  
Philadelphia Chromosome ◽  
Myeloproliferative Neoplasm ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Thai Population ◽  
Specific Pcr ◽  
Allele Specific ◽  
Calr Mutations

Background: Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms (MPN) are a group of hematological malignancies, including polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF). Mutations of JAK2, CALR, MPL, and ASXL1 are associated with carcinogenesis and clinical characteristics of Ph-negative MPN. However, the availability of the data regarding these mutations is relatively limited in Thai population. Objective: To investigate these mutations in Thai Ph-negative MPN patients. Materials and Methods: One hundred thirty-six MPN (48 PV, 72 ET, and 16 PMF) cases were enrolled. Mutations of JAK2 V617F and MPL W515L/K mutations were investigated using allele-specific PCR (AS-PCR) and confirmed by sequencing. CALR and ASXL1 mutations were investigated using Sanger sequencing. Results: The JAK2 V617F mutation was detected in 83.3% of PV, 66.6% of ET, and 50.0% of PMF, and correlated with higher RBC, WBC, and PLT counts in PV. CALR mutations were detected in 16.7% of ET and 12.5% of PMF and associated with a higher PLT count in ET. The MPL W515L mutation was detected in one PMF patient. ASXL1 mutations were detected in 6.3% of PV, 8.3% of ET, and 12.4% of PMF, with c.1954G>A being the preponderant mutational form. ASXL1 mutations increased the risk (RR 27.6) and accelerated the onset of AML transformation. Conclusion: The present study provided the prevalence and clinical correlation of JAK2, CALR, MPL, and ASXL1 mutations among Thai Ph-negative MPN patients. The association of ASXL1 mutations with adverse clinical outcomes suggested the potential usefulness of these mutations as a prognostic marker for Ph-negative MPN patients. Keywords: JAK2, MPL, CALR, ASXL1, Thai, Philadelphia-negative myeloproliferative neoplasm (MPN)

scholarly journals Warfarin Genotyping in a Single PCR Reaction for Microchip Electrophoresis

2012 ◽  
Vol 58 (4) ◽  
pp. 725-731 ◽  
Author(s):  
Brian L Poe ◽  
Doris M Haverstick ◽  
James P Landers
Keyword(s):  
Turnaround Time ◽  
Microchip Electrophoresis ◽  
Amplification Refractory Mutation System ◽  
Sample Population ◽  
Nucleotide Polymorphisms ◽  
Specific Pcr ◽  
Mutation System ◽  
Pcr Products ◽  
Allele Specific ◽  
Cytochrome P450 Family

Abstract BACKGROUND Warfarin is the most commonly prescribed oral anticoagulant medication but also is the second leading cause of emergency room visits for adverse drug reactions. Genetic testing for warfarin sensitivity may reduce hospitalization rates, but prospective genotyping is impeded in part by the turnaround time and costs of genotyping. Microfluidics-based assays can reduce reagent consumption and analysis time; however, no current assay has integrated multiplexed allele-specific PCR for warfarin genotyping with electrophoretic microfluidics hardware. Ideally, such an assay would use a single PCR reaction and, without further processing, a single microchip electrophoresis (ME) run to determine the 3 single-nucleotide polymorphisms (SNPs) affecting warfarin sensitivity [i.e., CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9) *2, CYP2C9 *3, and the VKORC1 (vitamin K epoxide reductase complex 1) A/B haplotype]. METHODS We designed and optimized primers for a fully multiplexed assay to examine 3 biallelic SNPs with the tetraprimer amplification refractory mutation system (T-ARMS). The assay was developed with conventional PCR equipment and demonstrated for microfluidic infrared-mediated PCR. Genotypes were determined by ME on the basis of the pattern of PCR products. RESULTS Thirty-five samples of human genomic DNA were analyzed with this multiplex T-ARMS assay, and 100% of the genotype determinations agreed with the results obtained by other validated methods. The sample population included several genotypes conferring warfarin sensitivity, with both homozygous and heterozygous genotypes for each SNP. Total analysis times for the PCR and ME were approximately 75 min (1-sample run) and 90 min (12-sample run). CONCLUSIONS This multiplexed T-ARMS assay coupled with microfluidics hardware constitutes a promising avenue for an inexpensive and rapid platform for warfarin genotyping.

scholarly journals COMPARISON OF DIFFERENT METHODS OF MOLECULAR-GENETIC ANALYSIS OF SOMATIC MUTATIONS IN K-RAS GENE IN PATIENTS WITH COLORECTAL CANCER

2012 ◽  
Vol 67 (2) ◽  
pp. 35-41 ◽  
Author(s):  
F. A. Amosenko ◽  
I. V. Karpov ◽  
A. V. Polyakov ◽  
S. P. Kovalenko ◽  
V. A. Shamanin ◽  
...  
Keyword(s):  
Molecular Genetic ◽  
False Negative ◽  
Point Mutations ◽  
Real Life ◽  
Molecular Genetic Analysis ◽  
Ras Gene ◽  
Specific Pcr ◽  
Specificity And Sensitivity ◽  
Allele Specific ◽  
Allele Specific Pcr

Two approaches to somatic point mutations in 12 and 13 codones of K-ras gene were analyzed: PCR/SSCP/AСRS/sequencing and allele-specific PCR in the real-life regimen (Russian set «KRAS-7M»). The comparison was carried out on 62 examples of genomic DNA extracted from frozen colon carcinomas, which underwent manual dissection. The results obtained in two attempts were consistent in 95,2% (N=59). Specificity and sensitivity of K-ras mutations detection using «KRAS-7M» set were 100 and 96,4% respectively, and 94,1 and 100% respectievly using PCR/SSCP/AСRS/ automatic sequencing. False positive results were absent when detecting with «KRAS-7M» and accounted for 2 cases (5,9%) when using PCR/SSCP/ AСRS/automatic sequencing. The only false negative response (3,6%) was obtained analyzing mutations using «KRAS-7M».

Simple and Rapid Detection of Factor V Leiden by Allele-specific PCR Amplification

1996 ◽  
Vol 75 (05) ◽  
pp. 757-759 ◽  
Author(s):  
Rainer Blasczyk ◽  
Markus Ritter ◽  
Christian Thiede ◽  
Jenny Wehling ◽  
Günter Hintz ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Pcr Amplification ◽  
Factor V ◽  
Specific Pcr ◽  
Pcr Rflp ◽  
Allele Specific ◽  
Specific Amplification ◽  
Allele Specific Pcr

SummaryResistance to activated protein C is the most common hereditary cause for thrombosis and significantly linked to factor V Leiden. In this study, primers were designed to identify the factor V mutation by allele-specific PCR amplification. 126 patients with thromboembolic events were analysed using this technique, PCR-RFLP and direct sequencing. The concordance between these techniques was 100%. In 27 patients a heterozygous factor VGln506 mutation was detected, whereas one patient with recurrent thromboembolism was homozygous for the point mutation. Due to its time- and cost-saving features allele-specific amplification should be considered for screening of factor VGln506.

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