The comparison of clinical features between JAK2V617F positive and negative subgroups of essential thrombocythemia

17523 Background: JAK2V617F is a novel molecular mechanisms of BCR/ABL-negative MPD especially polycythemia vera. However, its role may not be clearly identified in essential thrombocythemia (ET). Methods: To investigate its prevalence and clinical significance in Chinese ET patients, Allele-Specific PCR (AS-PCR) in combination with sequence analysis were introduced to screen JAK2V617F mutation in our research. Genomic DNA of 40 ET patients was extracted from peripheral blood mononuclear cells, and AS-PCR was firstly adopted to amplify the exon 12 of JAK2 gene which harbored V617F mutation. The positive samples with JAK2V617F mutation were further confirmed by sequence analysis. In addition, the features of JAK2V617F positive and negative subgroup were compared according to laboratory examination. Results: The JAK2V617F mutation was identified in 18 ET patients (45%, 18/40) by AS-PCR and sequence analysis. Its presence is significantly associated with a higher hemoglobin, hematocrit and neutrophilic granulocyte percentage in ET patients. Conclusions: AS-PCR is a sensitive and accurate technique in identification of JAK2V617F mutation. JAK2V617F mutation is of important significance in Chinese ET patients. Moreover, this ET subgroup may be prone to transform to polycythemia vera. No significant financial relationships to disclose.

Download Full-text

The clinical implications of JAK2V617F mutation in idiopathic myelofibrosis

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.17542 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 17542-17542

Author(s):

W. Li ◽

S. Zhang ◽

J. Li

Keyword(s):

Sequence Analysis ◽

Molecular Mechanisms ◽

Mononuclear Cells ◽

Clinical Implications ◽

Idiopathic Myelofibrosis ◽

Peripheral Blood Mononuclear ◽

Clinical Process ◽

Specific Pcr ◽

Allele Specific ◽

Allele Specific Pcr

17542 Background: JAK2V617F is a novel molecular mechanisms of BCR/ABL-negative MPD especially polycythemia vera. However, its role may not be well clarified in idiopathic myelofibrosis (IMF). Methods: To investigate its prevalence and clinical significance in Chinese IMF patients, Allele-Specific PCR (AS-PCR) in combination with sequence analysis were introduced to screen JAK2V617F mutation in our research. Genomic DNA of 12 IMF patients was extracted from peripheral blood mononuclear cells, and AS-PCR was firstly adopted to amplify the exon 12 of JAK2 gene which harbored V617F mutation. The positive samples with JAK2V617F mutation were further confirmed by sequence analysis. In addition, the clinical features of JAK2V617F positive and negative subgroup were compared. Results: The JAK2V617F mutation was identified in 6 IMF patients (50%, 6/12) by AS-PCR and sequence analysis. Its presence is significantly associated with a higher platelet counts, megakaryocyte counts of bone marrow and incidence rate of thrombosis in IMF patients. Conclusions: AS-PCR is a sensitive and accurate technique in identification of JAK2V617F mutation. JAK2V617F mutation of our research is similar to other reports. Moreover, the clinical process of JAK2V617F positive subgroup may be more invasive in comparison with JAK2V617F negative subgroup. No significant financial relationships to disclose.

Download Full-text

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

Blood ◽

10.1182/blood.v110.11.2533.2533 ◽

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

Download Full-text

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650362 ◽

1996 ◽

Vol 75 (05) ◽

pp. 757-759 ◽

Cited By ~ 11

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.

Download Full-text

MUPrimer : a tool for finding allele specific PCR-primers for homologous gene sequences

10.32469/10355/6660 ◽

2009 ◽

Author(s):

M. Mursaleen Ahmad

Keyword(s):

Pcr Primers ◽

Homologous Gene ◽

Gene Sequences ◽

Specific Pcr ◽

Allele Specific ◽

Allele Specific Pcr

Download Full-text

Development of genic KASP SNP markers from RNA-Seq data for map-based cloning and marker-assisted selection in maize

BMC Plant Biology ◽

10.1186/s12870-021-02932-8 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Zhengjie Chen ◽

Dengguo Tang ◽

Jixing Ni ◽

Peng Li ◽

Le Wang ◽

...

Keyword(s):

Marker Assisted Selection ◽

Inbred Lines ◽

Average Density ◽

Snp Markers ◽

Data Sets ◽

Rna Seq ◽

Specific Pcr ◽

Maize Inbred Lines ◽

Allele Specific ◽

Allele Specific Pcr

Abstract Background Maize is one of the most important field crops in the world. Most of the key agronomic traits, including yield traits and plant architecture traits, are quantitative. Fine mapping of genes/ quantitative trait loci (QTL) influencing a key trait is essential for marker-assisted selection (MAS) in maize breeding. However, the SNP markers with high density and high polymorphism are lacking, especially kompetitive allele specific PCR (KASP) SNP markers that can be used for automatic genotyping. To date, a large volume of sequencing data has been produced by the next generation sequencing technology, which provides a good pool of SNP loci for development of SNP markers. In this study, we carried out a multi-step screening method to identify kompetitive allele specific PCR (KASP) SNP markers based on the RNA-Seq data sets of 368 maize inbred lines. Results A total of 2,948,985 SNPs were identified in the high-throughput RNA-Seq data sets with the average density of 1.4 SNP/kb. Of these, 71,311 KASP SNP markers (the average density of 34 KASP SNP/Mb) were developed based on the strict criteria: unique genomic region, bi-allelic, polymorphism information content (PIC) value ≥0.4, and conserved primer sequences, and were mapped on 16,161 genes. These 16,161 genes were annotated to 52 gene ontology (GO) terms, including most of primary and secondary metabolic pathways. Subsequently, the 50 KASP SNP markers with the PIC values ranging from 0.14 to 0.5 in 368 RNA-Seq data sets and with polymorphism between the maize inbred lines 1212 and B73 in in silico analysis were selected to experimentally validate the accuracy and polymorphism of SNPs, resulted in 46 SNPs (92.00%) showed polymorphism between the maize inbred lines 1212 and B73. Moreover, these 46 polymorphic SNPs were utilized to genotype the other 20 maize inbred lines, with all 46 SNPs showing polymorphism in the 20 maize inbred lines, and the PIC value of each SNP was 0.11 to 0.50 with an average of 0.35. The results suggested that the KASP SNP markers developed in this study were accurate and polymorphic. Conclusions These high-density polymorphic KASP SNP markers will be a valuable resource for map-based cloning of QTL/genes and marker-assisted selection in maize. Furthermore, the method used to develop SNP markers in maize can also be applied in other species.

Download Full-text

Two User-Friendly Molecular Markers Developed for the Identification of Hybrid Lethality Genes in Brassica oleracea

Agronomy ◽

10.3390/agronomy11050982 ◽

2021 ◽

Vol 11 (5) ◽

pp. 982

Author(s):

Zhiliang Xiao ◽

Congcong Kong ◽

Fengqing Han ◽

Limei Yang ◽

Mu Zhuang ◽

...

Keyword(s):

Molecular Markers ◽

Brassica Oleracea ◽

Rapid Identification ◽

Inbred Lines ◽

Hybrid Lethality ◽

Specific Pcr ◽

Allele Specific ◽

User Friendly ◽

Allele Specific Pcr ◽

Kasp Marker

Cabbage (Brassica oleracea) is an important vegetable crop that is cultivated worldwide. Previously, we reported the identification of two dominant complementary hybrid lethality (HL) genes in cabbage that could result in the death of hybrids. To avoid such losses in the breeding process, we attempted to develop molecular markers to identify HL lines. Among 54 previous mapping markers closely linked to BoHL1 or BoHL2, only six markers for BoHL2 were available in eight cabbage lines (two BoHL1 lines; three BoHL2 lines; three lines without BoHL); however, they were neither universal nor user-friendly in more inbred lines. To develop more accurate markers, these cabbage lines were resequenced at an ~20× depth to obtain more nucleotide variations in the mapping regions. Then, an InDel in BoHL1 and a single-nucleotide polymorphism (SNP) in BoHL2 were identified, and the corresponding InDel marker MBoHL1 and the competitive allele-specific PCR (KASP) marker KBoHL2 were developed and showed 100% accuracy in eight inbred lines. Moreover, we identified 138 cabbage lines using the two markers, among which one inbred line carried BoHL1 and 11 inbred lines carried BoHL2. All of the lethal line genotypes obtained with the two markers matched the phenotype. Two markers were highly reliable for the rapid identification of HL genes in cabbage.

Download Full-text