Dataset Information

Detection of CALR Mutations Using High Resolution Melting Curve Analysis (HRM-A); Application on a Large Cohort of Greek ET and MF Patients.

ABSTRACT: Background and Objectives:Somatic mutations in the calreticulin gene (CALR) are detected in approximately 70% of patients with essential thrombocythemia (ET) and primary or secondary myelofibrosis (MF), lacking the JAK2 and MPL mutations. To determine the prevalence of CALR frameshift mutations in a population of MPN patients of Greek origin, we developed a rapid low-budget PCR-based assay and screened samples from 5 tertiary Haematology units. This is a first of its kind report of the Greek patient population that also disclosed novel CALR mutants. Methods:MPN patient samples were collected from different clinical units and screened for JAK2 and MPL mutations after informed consent was obtained. Negative samples were analyzed for the presence of CALR mutations. To this end, we developed a modified post Real Time PCR High-Resolution Melting Curve analysis (HRM-A) protocol. Samples were subsequently confirmed by Sanger sequencing. Results:Using this protocol we screened 173 MPN, JAK2 and MPL mutation negative, patients of Greek origin, of whom 117 (67.63%) displayed a CALR exon nine mutation. More specifically, mutations were detected in 90 out of 130 (69.23%) essential thrombocythaemia cases (ET), in 18 out of 33 (54.55%) primary myelofibrosis patients (pMF) and in 9 out of 10 (90%) cases of myelofibrosis secondary to ET (post-ET sMF). False positive results were not detected. The limit of detection (LoD) of our protocol was 2%. Furthermore, our study revealed six rare novel mutations which are to be added in the COSMIC database. Conclusions:Overall, our method could rapidly and cost-effectively detect the mutation status in a representative cohort of Greek patients; the mutation make-up in our group was not different from what has been published for other national groups.

SUBMITTER: Giannopoulos A

PROVIDER: S-EPMC6328041 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Detection of CALR Mutations Using High Resolution Melting Curve Analysis (HRM-A); Application on a Large Cohort of Greek ET and MF Patients.

Giannopoulos Andreas A Rougkala Niki N Loupis Theodoros T Mantzourani Marina M Viniou Nora-Athina NA Variami Eleni E Vassilakopoulos Theodoros P TP Dryllis George G Kotsianidis Ioannis I Gougopoulou Theodora T Politou Marianna M Konstantopoulos Kostas K Vassilopoulos George G

Mediterranean journal of hematology and infectious diseases 20190101 1

<h4>Background and objectives</h4>Somatic mutations in the calreticulin gene (CALR) are detected in approximately 70% of patients with essential thrombocythemia (ET) and primary or secondary myelofibrosis (MF), lacking the JAK2 and MPL mutations. To determine the prevalence of CALR frameshift mutations in a population of MPN patients of Greek origin, we developed a rapid low-budget PCR-based assay and screened samples from 5 tertiary Haematology units. This is a first ...[more]

PMID: 30671215

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Project description:BackgroundEarthworm communities are an important component of soil biodiversity and contribute to a number of ecosystem functions such as soil-nutrient cycling. Taxonomic identification is an essential requirement to assess earthworm biodiversity and functionality. Although morphological identification of species is labour-intensive, it is the most commonly used method due to a lack of cost-efficient alternatives. Molecular approaches to identify earthworms at species and haplotype level such as DNA barcoding are gaining popularity in science but are rarely applied in practice. In contrast to barcoding, the differentiation of PCR products based on their thermal denaturation properties using high-resolution melting (HRM) curve analysis is a fast and cost-efficient molecular closed-tube, post-PCR tool that allows identification of taxa.MethodsWe developed a HRM curve assay to identify eight earthworm species common to agricultural soils in Central Europe (Allolobophora chlorotica, Aporrectodea caliginosa, Apo. limicola, Apo. longa, Apo. rosea, Lumbricus castaneus, L. rubellus, and L. terrestris). For this, a new primer pair targeting a 158-bp long subregion of the cytochrome c oxidase I (COI) gene was designed. Our HRM assay was further tested for the differentiation of COI haplotypes using 28 individuals of the earthworm species Allo. chlorotica. Furthermore, we developed a novel extraction method for DNA from earthworm tissue that is fast and requires minimal consumables and laboratory equipment.ResultsThe developed HRM curve assay allowed identifying all eight earthworm species. Performing the assay on 28 individuals of the earthworm species Allo. chlorotica enabled the distinction among different COI haplotypes. Furthermore, we successfully developed a rapid, robust, scalable, and inexpensive method for the extraction of earthworm DNA from fresh or frozen tissue.ConclusionsHRM curve analysis of COI genes has the potential to identify earthworm species and haplotypes and could complement morphological identification, especially for juvenile or damaged individuals. Our rapid and inexpensive DNA extraction method from earthworm tissue helps to reduce the costs of molecular analyses and thereby promote their application in practice.

Dataset Information

Detection of CALR Mutations Using High Resolution Melting Curve Analysis (HRM-A); Application on a Large Cohort of Greek ET and MF Patients.

Publications

Detection of <i>CALR</i> Mutations Using High Resolution Melting Curve Analysis (HRM-A); Application on a Large Cohort of Greek ET and MF Patients.

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