Contributions
Abstract: PB1700
Type: Publication Only
Session title: Myeloproliferative neoplasms - Clinical
Background
In accordance with the WHO clinical guidelines, the analysis of somatic mutations in the CALR gene, as well as mutations in the JAK2 and MPL genes, are included in the list of criteria for the Ph-myeloproliferative neoplasms (Ph-MPN) diagnosis. More than 50 different mutation variants have been found in the CALR gene, among which the most frequent are a 52 bp deletion (p.L367fs*46), also called type 1, and a 5 bp insertion (p.K385fs*47), also called type 2 (88%). The remaining 12% are other type less frequent indels or combinations. Therefore, it is important to develop an inexpensive screening test that can detect any mutations in the analyzed DNA fragment of the CALR gene.
Aims
The aim of this study was to screen CALR mutation by HRM analysis using the CFX96 thermocycler and the Precision Melt Analysis software (Bio-Rad, USA) for patients with Ph-MPN.
Methods
5 CALR«+» DNA samples from MPN patients were included in this study. HRM analysis was performed using a Precision Melt Supermix reagent kit in the presence of Eva Green dye (Bio-Rad, USA). PCR with an additional high-resolution melting step was carried out on a CFX96 device (Bio-Rad, USA) according to the following program: denaturation at 95 ° C for 2 minutes, then 40 cycles at 95 ° C for 10 seconds, 57 ° C in for 30 seconds and 72 ° C for 30 seconds. The high resolution melting program consisted of denaturation at 95 ° C for 30 seconds, renaturation at 60 ° C for 1 minute, and melting at 65 ° C to 95 ° C with a 0.2 ° C gradient in 10 seconds. For the most common CALR mutations (c.1092_1143del and c.1154_1155insTTGTC), a threshold determination of the mutant allele presence was analyzed. To analyze the threshold for determining the proportion of the mutant allele, dilution of cloned wild-type and mutated samples was performed to obtain samples with different levels of allelic loads 100%, 50%, 25%, 12.5%, 6.25%, 3.13%
Results
The results of CALR«+» samples analysis after processing in the 'Precision Melt Analysis' program are shown in Figure 1. According to the figure, all melting curves of amplification products from CALR«+» DNA samples are clearly divided into two clusters relative to samples without mutations (cluster №1). So, two out of five CALR«+» samples (the sample with a classical mutation of type 2 (c.1154_1155insTTGTC) and the sample with a combined mutation (c.1128_1129insCTTTGCT; c.1131_1133delAGA)) were assigned to one cluster (cluster №2). The three other samples with type 1 mutation (c.1092_1143del) were assigned also to one cluster (cluster №3). The detection thresholds in case of the c.1092_1143del and c.1154_1155insTTGTC mutation analysis are 12.5% of the presence of the mutant allele in the samples.
Conclusion
Therefore, the HRM analysis that was conducted on the CFX96 allows screening of highly specific mutation for the diagnosis of Ph-MPN in the exon 9 of the CALR gene. The inclusion of this screening research in the laboratory testing algorithm improves the efficiency and accessibility of molecular genetic technologies in the diagnosis of Ph-MPN.
Keyword(s): Myeloproliferative disorder, Somatic mutation
Abstract: PB1700
Type: Publication Only
Session title: Myeloproliferative neoplasms - Clinical
Background
In accordance with the WHO clinical guidelines, the analysis of somatic mutations in the CALR gene, as well as mutations in the JAK2 and MPL genes, are included in the list of criteria for the Ph-myeloproliferative neoplasms (Ph-MPN) diagnosis. More than 50 different mutation variants have been found in the CALR gene, among which the most frequent are a 52 bp deletion (p.L367fs*46), also called type 1, and a 5 bp insertion (p.K385fs*47), also called type 2 (88%). The remaining 12% are other type less frequent indels or combinations. Therefore, it is important to develop an inexpensive screening test that can detect any mutations in the analyzed DNA fragment of the CALR gene.
Aims
The aim of this study was to screen CALR mutation by HRM analysis using the CFX96 thermocycler and the Precision Melt Analysis software (Bio-Rad, USA) for patients with Ph-MPN.
Methods
5 CALR«+» DNA samples from MPN patients were included in this study. HRM analysis was performed using a Precision Melt Supermix reagent kit in the presence of Eva Green dye (Bio-Rad, USA). PCR with an additional high-resolution melting step was carried out on a CFX96 device (Bio-Rad, USA) according to the following program: denaturation at 95 ° C for 2 minutes, then 40 cycles at 95 ° C for 10 seconds, 57 ° C in for 30 seconds and 72 ° C for 30 seconds. The high resolution melting program consisted of denaturation at 95 ° C for 30 seconds, renaturation at 60 ° C for 1 minute, and melting at 65 ° C to 95 ° C with a 0.2 ° C gradient in 10 seconds. For the most common CALR mutations (c.1092_1143del and c.1154_1155insTTGTC), a threshold determination of the mutant allele presence was analyzed. To analyze the threshold for determining the proportion of the mutant allele, dilution of cloned wild-type and mutated samples was performed to obtain samples with different levels of allelic loads 100%, 50%, 25%, 12.5%, 6.25%, 3.13%
Results
The results of CALR«+» samples analysis after processing in the 'Precision Melt Analysis' program are shown in Figure 1. According to the figure, all melting curves of amplification products from CALR«+» DNA samples are clearly divided into two clusters relative to samples without mutations (cluster №1). So, two out of five CALR«+» samples (the sample with a classical mutation of type 2 (c.1154_1155insTTGTC) and the sample with a combined mutation (c.1128_1129insCTTTGCT; c.1131_1133delAGA)) were assigned to one cluster (cluster №2). The three other samples with type 1 mutation (c.1092_1143del) were assigned also to one cluster (cluster №3). The detection thresholds in case of the c.1092_1143del and c.1154_1155insTTGTC mutation analysis are 12.5% of the presence of the mutant allele in the samples.
Conclusion
Therefore, the HRM analysis that was conducted on the CFX96 allows screening of highly specific mutation for the diagnosis of Ph-MPN in the exon 9 of the CALR gene. The inclusion of this screening research in the laboratory testing algorithm improves the efficiency and accessibility of molecular genetic technologies in the diagnosis of Ph-MPN.
Keyword(s): Myeloproliferative disorder, Somatic mutation