Development of V617F JAK2 Associated Myeloproliferative Neoplasms Is a Non-Random Event That Is Strongly Dependent on JAK2 Haplotype

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 173-173
Author(s):  
Nicholas C.P. Cross ◽  
Amy V. Jones ◽  
Richard T. Silver ◽  
David Oscier ◽  
Georgia Metzgeroth ◽  
...  
Keyword(s):  
Myeloproliferative Neoplasms ◽  
Epidemiological Data ◽  
Snp Analysis ◽  
Nucleotide Polymorphisms ◽  
Wild Type ◽  
Exact Test ◽  
Specific Pcr ◽  
Core Haplotype ◽  
Functional Variant ◽  
Allele Specific

Abstract Epidemiological data and family studies have indicated that inherited factors may predispose to the development of myeloproliferative neoplasms (MPN). It has also been suggested that single nucleotide polymorphisms (SNPs) within JAK2 are associated with specific MPN subtypes. To explore the role of inherited factors in more detail, we initially performed quantitative analysis of a series of JAK2 SNPs in homozygous PV cases (%V617F >50%; n=73). Most mutant haplotypes could be read directly from the distorted allele ratios brought about by expansion of the homozygous clone. In many cases with 50–90% V617F, the residual wild type haplotype could also be read. Strikingly, of the 144 V617F alleles that could be determined, 111 (77%) had an identical core haplotype (subsequently designated 46/1) whereas only 9/76 (12%) residual wild type alleles were 46/1 (P = 1.9e-21, Fisher’s exact test). To explore this observation in more detail we first determined the haplotype structure of JAK2 using 14 SNPs genotyped by the Wellcome Trust Case Control Consortium (WTCCC) in 1500 UK blood donors. Nine haplotypes were inferred using the program PHASE that accounted for 94% of alleles, with a frequency of 0.24 for haplotype 46/1. Haplotype inference and tag SNP analysis revealed that 46/1 was also more frequent in heterozygous V617F positive MPD cases (135/354 alleles) compared to 188 locally sourced healthy controls (92/376 alleles; P = 0.0001) as well as the WTCCC cohort (P = 3.3e-8). Haplotype 46/1 was more frequent in all V617F positive disease entities compared to controls: PV (n=203; P=1.2e-16), ET (n=81; P=1.2e-9) and MF (n=41; P=8.0e-5) however there was no difference in the frequency of 46/1 between controls and V617F negative MPD / idiopathic erythrocytosis (n=123). To determine if heterozygous V617F also preferentially arose on a 46/1 allele as seen for homozygous cases, we developed an allele specific PCR between V617F and a SNP that tags this haplotype. In an analysis of 67 informative heterozygous V617F cases, 50 V617F alleles were 46/1 compared to only 17 residual wild type alleles (P=9.4e-9). We conclude that the 46/1 JAK2 haplotype is a strong predisposition factor for development of V617F associated MPDs (RR=2.6; 95% CI 2.3–2.9). The reason for this predisposition is currently unknown but it is likely that 46/1 is in linkage disequilibrium with an unknown constitutional functional variant that interacts with V617F JAK2.

scholarly journals Fine Mapping of qd1, a Dominant Gene that Regulates Stem Elongation in Bread Wheat

2021 ◽  
Vol 12 ◽  
Author(s):  
Yongdun Xie ◽  
Weiwei Zeng ◽  
Chaojie Wang ◽  
Daxing Xu ◽  
Huijun Guo ◽  
...  
Keyword(s):  
Bread Wheat ◽  
Stem Elongation ◽  
Dominant Gene ◽  
Nucleotide Polymorphisms ◽  
Sequencing Data ◽  
Specific Pcr ◽  
Yield Determination ◽  
Allele Specific ◽  
Stem Development ◽  
Kasp Markers

Stem elongation is a critical phase for yield determination and, as a major trait, is targeted for manipulation for improvement in bread wheat (Triticum aestivum L.). In a previous study, we characterized a mutant showing rapid stem elongation but with no effect on plant height at maturity. The present study aimed to finely map the underlying mutated gene, qd1, in this mutant. By analyzing an F2 segregating population consisting of 606 individuals, we found that the qd1 gene behaved in a dominant manner. Moreover, by using the bulked segregant RNA sequencing (BSR-seq)-based linkage analysis method, we initially mapped the qd1 gene to a 13.55 Mb region on chromosome 4B (from 15.41 to 28.96 Mb). This result was further confirmed in F2 and BC3F2 segregating populations. Furthermore, by using transcriptome sequencing data, we developed 14 Kompetitive Allele-Specific PCR (KASP) markers and then mapped the qd1 gene to a smaller and more precise 5.08 Mb interval from 26.80 to 31.88 Mb. To develop additional markers to finely map the qd1 gene, a total of 4,481 single-nucleotide polymorphisms (SNPs) within the 5.08 Mb interval were screened, and 25 KASP markers were developed based on 10x-depth genome resequencing data from both wild-type (WT) and mutant plants. The qd1 gene was finally mapped to a 1.33 Mb interval from 28.86 to 30.19 Mb on chromosome 4B. Four candidate genes were identified in this region. Among them, the expression pattern of only TraesCS4B02G042300 in the stems was concurrent with the stem development of the mutant and WT. The qd1 gene could be used in conjunction with molecular markers to manipulate stem development in the future.

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

Diagnostics ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 872
Author(s):  
Alexey S. Chubarov ◽  
Igor P. Oscorbin ◽  
Maxim L. Filipenko ◽  
Alexander A. Lomzov ◽  
Dmitrii V. Pyshnyi
Keyword(s):  
Kras Mutation ◽  
Mutation Detection ◽  
Synergetic Effect ◽  
Model Systems ◽  
Wild Type ◽  
Kras Mutations ◽  
Specific Pcr ◽  
Mutational Status ◽  
Allele Specific ◽  
Pcr Method

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.

Detection of Imatinib-Resistant BCR-ABL Mutants in Drug-Naïve Patients: Correlation with Disease Phase and Clonal Evolution but Not with Response to Treatment.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 273-273
Author(s):  
Michael W. Deininger ◽  
Stephanie Willis ◽  
Thoralf Lange ◽  
Shannon McWeeney ◽  
Sandra Otto ◽  
...  
Keyword(s):  
Mutation Detection ◽  
Clonal Evolution ◽  
Drug Binding ◽  
Disease Stage ◽  
Wild Type ◽  
Imatinib Resistance ◽  
Specific Pcr ◽  
Blastic Phase ◽  
Allele Specific ◽  
Allele Specific Pcr

Abstract Background: Point mutations in the kinase domain (KD) of BCR-ABL that impair drug binding are a common cause of imatinib resistance. In some cases the mutations present at relapse were detected in pretherapeutic samples, suggesting selection in the presence of imatinib and a possible association with disease progression. To test this hypothesis, we studied the presence of KD mutations in imatinib-naïve patients in various disease phases. Methods and patients: we developed fluorescent allele-specific RT-PCR assays for 8 common KD mutations (Q252H, Y253F, Y253H, E255K, E255V, T315I, M351T, F359V), comprising approximately 75% of mutations detected in patients, using universal BCR and mutation-specific ABL primers. Mutation-complementary oligonucleotides were further modified to favor amplification of mutant over wild type. Sensitivity and specificity were optimized in serial dilutions of mutant in wild type, using plasmid DNA or cDNA extracted from BaF3 cells expressing BCR-ABL constructs. The assays reproducibly detected mutations with a sensitivity of at least 10−4 – 10−5. To avoid false-positive results cDNA was prepared in a physically separate lab that never handled recombinant mutant BCR-ABL. Negative controls included “blanks”, K562 cells (positive for wild type BCR-ABL) and HL60 cells (BCR-ABL-negative). Pretherapeutic samples from 20 patients in first chronic, 24 in accelerated and 21 in blastic phase were studied. Results: Mutations were detected in 13 samples from blastic phase (5) or from accelerated phase (8) but in none from chronic phase. Mutations were: T315I (7), Y253F (2), 253H (2), Q252H (1) and F359V (1). Sequencing of the same material detected F359V in one sample (consistent with the allele specific PCR), and K247R in another (positive for T315I by allele-specific PCR) but was wild type in the remaining 10 samples with good quality readings. Multivariate analysis of baseline factors revealed significant and independent correlations between mutation detection and disease phase (P = 0.0008) and clonal cytogenetic evolution (P = 0.0004). In contrast, mutation detection was not an independent predictor of failure to achieve complete hematologic response (CHR) or major cytogenetic response (MCR) and was not correlated with overall survival. Four patients with mutations achieved CHR and 3 MCR; 4 patients failed to achieve CHR, 1 died from neutropenic sepsis and 1 discontinued because of skin toxicity. Thus far, follow-up specimens during imatinib therapy have been sequenced in 6 patients. In 2 cases (T315I and Q252H) the identical mutation was detected, one case (T315I) showed K247R (detected prior to therapy by sequencing but not included in the allele specific assays), and 3 cases were wild type BCR-ABL. Conclusion: We provide direct evidence that the presence of BCR-ABL KD mutantions is correlated with disease stage. Moreover, the strong association with clonal evolution suggests that genomic instability may be responsible for both phenomena. However, not all mutants that were detected prior to therapy were subsequently selected in the presence of imatinib, suggesting that some mutations may occur in cells without self-renewal capacity or that additional factors may be required to confer a fully resistant phenotype.

Novel Assay for the Identification of NOTCH1 PEST Domain Mutations Using Fragment Analysis and Allele Specific PCR

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5127-5127
Author(s):  
Paulo Vidal Campregher ◽  
Roberta Cardoso Petroni ◽  
Nair Muto ◽  
Rubia Santana ◽  
Roberta Sitnik ◽  
...  
Keyword(s):  
Cost Effective ◽  
Single Mutation ◽  
Wild Type ◽  
Double Peak ◽  
Fragment Analysis ◽  
Specific Pcr ◽  
Routine Identification ◽  
Allele Specific ◽  
Allele Specific Pcr ◽  
Notch1 Mutations

Abstract Abstract 5127 NOTCH1 is a proto-oncogene with activating mutations described in a variety of malignancies, including acute lymphoblastic leukemia (ALL), mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL). While the prognostic significance of NOTCH1 mutations remains controversial in ALL, recent data suggest that NOTCH1 PEST domain mutations are associated with adverse prognosis in patients with CLL. NOTCH1 mutations are found in around 8% of CLL patients at diagnosis and more than 30% of patients with advanced disease. Since this disease has a heterogeneous clinical course and few prognostic markers, we aimed at designing a fast, cost effective and robust assay to detect NOTCH1 PEST domain mutations in patients with CLL for the clinical laboratory. While 92% of the mutations in NOTCH1 PEST domain found in CLL are insertions or deletions, only 8% are represented by point mutations. Therefore we decided to use a fragment analysis approach in our assay. Given that a single mutation (c. 7544_7545delCT), represents roughly 75% of all PEST domain mutations in CLL we designed a test that can, at the same time, detect the presence of this mutation specifically and also any insertion or deletion in exon 34. We designed a PCR reaction using one FAM-labeled forward primer anchored at codon 2407 and two reverse primers. One specific for the c. 7544_7545delCT mutation anchored at codon 2414 yielding a product of 356 base pairs (bp) and one anchored at codon 2425, yielding a product of 391 bp, comprising the hot spot for mutations in the NOTCH1 PEST domain. Primers were designed with Primer3 software (http://frodo.wi.mit.edu/) and the specificity of the reaction evaluated using the tool “PCR in silico” (http://genome.ucsc.edu/cgi-bin/hgPcr?command=start). The test yields three possible outputs: A single 391 bp peak: wild type samplesThree peaks (391 bp, 389 bp and 356 bp): heterozygous for c. 7544_7545delCTTwo peaks (391 bp and another bigger or smaller, depending on the size of insertion/deletion): another insertion or deletion, but not c. 7544_7545delCT. We have studied 46 de-identified blood samples from patients with CLL, in several diverse stages, using our assay. In 40 patients, there was no NOTCH1 mutation detected. Six patients had a pattern compatible with c. 7544_7545delCT NOTCH1 mutation (see figure 1), and no patients presented with another mutation. Overall the frequency of NOTCH1 mutations in our series was 13 %. Selected mutated samples were confirmed through amplicon sequencing. In conclusion, we have designed a robust, fast and cost effective assay for routine identification of NOTCH1 PEST domain mutations using fragment analysis and allele specific pcr that is suitable for implementation in the clinical setting for CLL patients evaluation. We will continue testing more CLL patients in order to identify another, rarer, NOTCH1 mutations. Figure 1. Assay Results for NOTCH1 PEST Domain Mutations A – Wild Type NOTCH1 revealed by the presence of a single 391 bp peak. B – Presence of heterozygous c. 7544_7545delCT mutation evidenced by the presence of a 356 bp peak, corresponding to the allele specific pcr peak; and a double peak at 391 bp and 389 bp positions, corresponding to the wild type product (391 bp) and to the mutated product (389 bp) detected with the wild type primers. Figure 1. Assay Results for NOTCH1 PEST Domain Mutations . / A – Wild Type NOTCH1 revealed by the presence of a single 391 bp peak. . / B – Presence of heterozygous c. 7544_7545delCT mutation evidenced by the presence of a 356 bp peak, corresponding to the allele specific pcr peak; and a double peak at 391 bp and 389 bp positions, corresponding to the wild type product (391 bp) and to the mutated product (389 bp) detected with the wild type primers. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Preventing Favism by Selecting Faba Bean Mutants Using Molecular Markers

2017 ◽  
Vol 3 (1) ◽  
pp. 2-6 ◽  
Author(s):  
Melody Song
Keyword(s):  
Faba Bean ◽  
Expressed Sequence Tag ◽  
Nucleotide Polymorphisms ◽  
Seed Selection ◽  
Single Nucleotide ◽  
Specific Pcr ◽  
Faba Beans ◽  
Allele Specific ◽  
Haemolytic Anemia ◽  
Allele Specific Pcr

Faba bean (Vicia faba) is an ancient legume species known for its high protein content. The usage and consumption of the faba bean is limited by a glycoside, vicine-convicine (VC). Consumption of VC causes haemolytic anemia in individuals with the genetic condition called favism. Faba beans with low VC concentration are opening the possibility of reduction of favism disease, but there are many challenges in analyzing VC concentration. The objective of this study was to develop expressed sequence tag (EST) markers that can differentiate between low VC content (LVC) and high VC content (HVC) faba bean genotypes. Three single nucleotide polymorphisms (SNPs) were discovered that distinguished between LVC and HVC genotypes. The SNPs were validated using Kompetitive Allele Specific PCR (KASP) and mass spectrometry phenotyping. Molecular marker SNP 316 (Intron of Medtr2g009270 at 1,851,012 bp) was the most successful marker in differentiating between LVC, HVC, and heterozygous faba bean genotypes. This marker has applications in seed selection and acceleration of breeding programs, which is the first step towards allowing all consumers concerned with the effects of favism to enjoy the nutritional value of faba bean.

scholarly journals A novel ultra-sensitive method for the detection of FGFR3 mutations in urine of bladder cancer patients – Design of the Urodiag® PCR Kit for surveillance of patients with non-muscle-invasive bladder cancer (NMIBC)

2020 ◽  
Author(s):  
Jean-Pierre ROPERCH ◽  
Claude HENNION
Keyword(s):  
Bladder Cancer ◽  
High Risk ◽  
Limit Of Detection ◽  
Cost Effective ◽  
Wild Type ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Multiplex Pcr Assay ◽  
Allele Specific ◽  
Fgfr3 Mutations

Abstract Background We have recently developed a highly accurate urine-based test, named Urodiag ® , associating FGFR3 mutation and DNA methylation assays for recurrence surveillance in patients with low-, intermediate-, and high-risk NMIBC. Previously, the detection of four FGFR3 mutations (G372C, R248C, S249C and Y375C) required amplification steps and PCR products were analyzed by capillary electrophoresis (Allele Specific-PCR, AS-PCR), which was expensive and time-consuming. Here, we present the development a novel ultra-sensitive multiplex PCR assay as called “Mutated Allele Specific Oligonucleotide-PCR (MASO-PCR)”, generating cost-effective, simple, fast and clinically applicable assay for the detection of FGFR3 mutations in voided urine. Methods Comparative clinical performances of MASO-PCR and AS-PCR technologies were performed from 263 urine DNA samples (87 FGFR3 mutated and 176 FGFR3 wild-type). In the development of Urodiag ® PCR Kit, we studied the stability and reproducibility of each all-in-one PCR master mix (single reaction mixture including all the necessary PCR components) for MASO-PCR and QM-MSPCR (Quantitative Multiplex Methylation-Specific PCR to co-amplify SEPTIN9 , HS3ST2 and SLIT2 methylated genes) assays. Results Complete concordance (100%) was observed between the MASO-PCR and AS-PCR results. Each PCR master mix displayed excellent reproducibility and stability after 12 months of storage at -20°C, with intra-assay standard deviations lower than 0.3 Ct and coefficient of variations (CV) lower than 1%. The limit of detection (LoD) of MASO-PCR was 5% mutant detection in a 95% of wild-type background. The limit of quantification (LoQ) of QM-MSPCR was 10 pg of bisulfite-converted DNA. Conclusions We developed and clinically validated the MASO-PCR assay, generating cost-effective, simple, fast and clinically applicable assay for the detection of FGFR3 mutations in urine. We also designed the Urodiag ® PCR Kit, which includes the MASO-PCR and QM-MSPCR assays. Adapted to routine clinical laboratory (simplicity, accuracy), the kit will be a great help to urologists for recurrence surveillance in patients at low-, intermediate- and high-risk NMIBC. Reducing the number of unnecessary cystoscopies, it will have extremely beneficial effects for patients (painless) and for the healthcare systems (low cost).

scholarly journals TNF-α -308G/A polymorphism and susceptibility to tuberculosis in Azeri population of Iran

Genetika ◽  
2016 ◽  
Vol 48 (3) ◽  
pp. 819-826 ◽  
Author(s):  
Sajjad Ghorghanlu ◽  
Mohammad Asgharzadeh ◽  
Hossein Samadi-Kafil ◽  
Fatemeh Khaki-Khatibi ◽  
Jalil Rashedi ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Tuberculosis Patient ◽  
P Value ◽  
Nucleotide Polymorphisms ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Tuberculosis Patients ◽  
Specific Pcr ◽  
Allele Specific ◽  
And Function

Single nucleotide polymorphisms (SNPs) in cytokine genes may alter the level and function of secreted cytokine; therefore, SNPs can influence the immune response. The aim of the present study was to determine the association of TNF-? -308G/A single nucleotide polymorphism in tuberculosis patients in the Azeri population of Iran. The TNF-308G/A single nucleotide polymorphism in the promoter region was genotyped by using the allele-specific PCR method in 200 healthy controls and 124 tuberculosis patients. The distribution of allele frequencies for TNF-? -308G/A polymorphism between control and tuberculosis patient groups was not significant (P-value = 0.058, OR = 1.5). Furthermore, no statistically significant association was found between TNF-? -308G/A genotype and resistance/susceptibility to TB (P-value = 0.102). Our results suggest that TNF-? -308G/A polymorphism has no measurable effect on the development of tuberculosis in Azeri population of Iran.

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