A novel molecular assay using hybridisation probes and melt curve analysis for<i>CALR</i>exon 9 mutation detection in myeloproliferative neoplasms

Keaney, Thomas A.; O’Connor, Louise; Krawczyk, Janusz; Abdelrahman, Moutaz A; Hayat, Amjad; Murray, Margaret; O’Dwyer, Michael; Percy, Melanie J.; Langabeer, S. E.; Haslam, Karl; Glynn, Barry; Mullen, Ciara; Keady, Evelyn; Lahiff, Sinéad; Smith, Terry J.

doi:10.1136/jclinpath-2016-204205

Cited by 2 publications

(4 citation statements)

References 22 publications

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“…5,11,14 Patients with JAK2 mutations had a higher WBC count, hemoglobin level, and thrombotic events, while patients with CALR mutations were younger and had a significantly higher platelet count. Some reports also demonstrated different clinical manifestations between type 1 and 2 CALR mutations; 10,[14][15] however, due to the small number of studied cases, no differences were shown in this study.…”

Section: Methodscontrasting

confidence: 71%

“…The distribution was different from other reports. 7,8,10,13,14,15 Recently, Keaney and Li et al, reported a lower incidence of type 1 mutation in myelofibrosis patients. 10,12 These data might suggest that there is a population difference in CALR mutations and highlight the need for further mutation survey outside type 1 and 2.…”

Section: Methodsmentioning

confidence: 99%

“…[5][6][7][8][9] It has diagnostic importance and also contributes to the clinical characteristics of ET patients; therefore, it is crucial to identify the mutations. [10][11][12][13][14][15] Although most of the mutations correspond to a 52 bp deletion (type 1) or a 5 bp insertion (type 2), there were more than 50 other mutations found. The variations of mutations continue to pose a challenge for a rapid and effective survey.…”

Section: Methodsmentioning

confidence: 99%

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Calreticulin mutation survey by high resolution melting method associated with unique presentations in essential thrombocythemic patients

Liu

Lee

Yeh

et al. 2020

Mediterr J Hematol Infect Dis

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Somatic mutations of exon 9 of calreticulin gene (CALR) were diagnosis and prognosis importance found in patients with JAK2V617F-negative essential thrombocythemia (ET). We survey CALR and JAK2 mutations in our ET patients and study the relationship between mutations and clinical presentations. A total of 60 ET patients were enrolled in the study, and CALR mutations were studied by high resolution melting (HRM) methods and sequencing in JAK2V617F-negative group retrospectively. Clinical manifestations were reviewed retrospectively from chart records. Twenty-one CALR mutations showed eight types of specific melting curves detected by the HRM method and sequencing validation among 26 JAK2 V617F-negative patients. Compared with JAK2 mutations, patients with CALR mutations were younger and had a higher platelet count, lower white cell counts, and lower hemoglobin levels significantly (p<0.05). From our study, HRM methods revealed unique curve types in screening for CALR mutations screening even for complicated mutations. The mutations can be identification rapidly, and cost-effectively by HRM method than other tools. The clinical presentations of CALR mutations from JAK2 mutations showed significant differences and should be checked in ET patients.

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Section: Methodscontrasting

confidence: 71%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Calreticulin mutation survey by high resolution melting method associated with unique presentations in essential thrombocythemic patients

Liu

Lee

Yeh

et al. 2020

Mediterr J Hematol Infect Dis

View full text Add to dashboard Cite

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“…In general, the treatment of MPNs using disease modifiers is further complicated, as the specific treatment depends on the individual mutation [ 16 ]. Diagnosis of MPNs is aided by the detection of the various associated mutations by polymerase chain reaction, sequencing, or other DNA-based methods [ 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 ], but the detection of disease-associated mutations may be facilitated or aided by mutation-specific immunohistochemistry (IHC) [ 31 , 32 , 33 ], methods which supplement each other. Moreover, antibodies such as these are invaluable reagents for studying the properties of CRT in relation to MPN.…”

Section: Introductionmentioning

confidence: 99%

Production and Characterization of Peptide Antibodies to the C-Terminal of Frameshifted Calreticulin Associated with Myeloproliferative Diseases

Mughal

Bergmann

Huynh

et al. 2022

IJMS

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Myeloproliferative Neoplasms (MPNs) constitute a group of rare blood cancers that are characterized by mutations in bone marrow stem cells leading to the overproduction of erythrocytes, leukocytes, and thrombocytes. Mutations in calreticulin (CRT) genes may initiate MPNs, causing a novel variable polybasic stretch terminating in a common C-terminal sequence in the frameshifted CRT (CRTfs) proteins. Peptide antibodies to the mutated C-terminal are important reagents for research in the molecular mechanisms of MPNs and for the development of new diagnostic assays and therapies. In this study, eight peptide antibodies targeting the C-terminal of CRTfs were produced and characterised by modified enzyme-linked immunosorbent assays using resin-bound peptides. The antibodies reacted to two epitopes: CREACLQGWTE for SSI-HYB 385-01, 385-02, 385-03, 385-04, 385-07, 385-08, and 385-09 and CLQGWT for SSI-HYB 385-06. For the majority of antibodies, the residues Cys1, Trp9, and Glu11 were essential for reactivity. SSI-HYB 385-06, with the highest affinity, recognised recombinant CRTfs produced in yeast and the MARIMO cell line expressing CRTfs when examined in Western immunoblotting. Moreover, SSI-HYB 385-06 occasionally reacted to CRTfs from MPN patients when analysed by flow cytometry. The characterized antibodies may be used to understand the role of CRTfs in the pathogenesis of MPNs and to design and develop new diagnostic assays and therapeutic targets.

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A novel molecular assay using hybridisation probes and melt curve analysis forCALRexon 9 mutation detection in myeloproliferative neoplasms

Abstract: The novel assay described has potential for application as a rapid, sensitive, high-throughput screening method in the clinical diagnostics setting.

Cited by 2 publications

References 22 publications

Calreticulin mutation survey by high resolution melting method associated with unique presentations in essential thrombocythemic patients

Calreticulin mutation survey by high resolution melting method associated with unique presentations in essential thrombocythemic patients

Production and Characterization of Peptide Antibodies to the C-Terminal of Frameshifted Calreticulin Associated with Myeloproliferative Diseases

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