PB2236 APPLICATION OF HETERODUPLEX ANALYSIS FOR CARL MUTATION DETECTION
Authors: Subbotina T. , Karnyushka A. , Dunaeva E. , Mironov K. , Vasiyliev E. , Mikhalev M. , Smelyanskaya M. , Garkusha T. , Khorzhevskyi V. 
Published: June 2019

Background:CARL exon 9 somatic mutations are highly specific to confirm the diagnosis of essential thrombocythemia (ET) and myelofibrosis (MF). Moreover, the cases of CALR mutations in the absence of JAK2 mutations when polycythemia vera (PV) or idiopathic erythrocytosis (IE) is diagnosed are reported in the literature. CALR mutations are concentrated in exon 9 and they involve various deletions and insertions as well as their combinations that lead to the frameshifting. Nowadays over 50 different CALR mutations associated with chronic myeloproliferative neoplasia (CMN) have been identified and classified. To identify all possible variants, it is necessary to use sequencing. However, due to the high cost of sequencing, developing a two‐stage algorithm for detect mutations in CARL exon 9 using inexpensive screening is of immediate practically necessity. We have previously proposed a two‐stage algorithm for detect mutations in JAK2 exon 12 using inexpensive screening test by heteroduplex analysis (Subbotina T et al, Haematologica 2017).Aims:The aim of this study was to demonstrate the feasibility of using heteroduplex analysis with separation of the PCR product by electrophoresis on non‐denaturing PAGE as the preliminary screening test for detection of CARL exon 9 somatic mutations.Methods:6 DNA samples of patients with different phenotypic form of CMN and different level of CARL mutation allele burden were included in this study. The informed consents from these patients were obtained. The presence of the CARL mutations and allele burden levels were identified by pyrosequencing method with PyroMark Q24 (Qiagen, Germany) and by Sanger's DNA sequencing with AB3500 (USA). The PCR with the additional stage of formation heteroduplexes was performed using the Real‐time PCR kit (Syntol, Russia) and CFX 96 Real Time System (Biorad, USA). PCR products were analyzed by electrophoresis in 8% PAGE.Results:The ET or MF diagnoses for five of six patients were confirmed by bone marrow trephine biopsies histological examination. It is important to note one out of six patients had a confirmed diagnosis of PV, which is a rare event. The results of electrophoresis on non‐denaturing PAGE are reported in Figure 1. All six patients have six different indel mutation variants in the 9 exon of the CARL: с. 1154_1155insGTGTC; с. 1128_1129insCTTTGCTT and c. 1131_1133delAGA; c.1100_1151del (52); c.1154_1155insTTGTC; c.1092_1143del (51); c.1154_1155insTTGTC, accordingly. The minimum number of inserted nucleotides were 5 and the maximum number of falling nucleotides were 52. Variants of CARL mutations for № 1, 4, 5 and 6 patients have been already described. Patients № 2 and № 3 carry new (yet included in the COSMIC website) mutations in CARL: с. 1128_1129insCTTTGCTT and c. 1131_1133delAGA; c.1100_1151del (52). The allele burden level ranged from 25% to 50%.Summary/Conclusion:The proposed variant of the heteroduplex analysis with separation of the PCR product by electrophoresis on non‐denaturing PAGE can be recommended for use as the preliminary screening test which is carried out before the confirming pyrosequencing or sanger's sequencing. The two‐stage approach allows to optimize the algorithm of the CARL exon 9 mutation detection and to improve the efficiency of testing for patients suspected of having CMN.image