Rare Case of Alternative Splicing Form Caused By L248V Mutation in CML Patient
Authors: Butylin Pavel , Matyuhina Natalia , Siordia Nadia , Lomaya Elza , Zaritskey Andrey 
Published: November 2019

Abstract
Clinical data: Subject: Male, 45 years old, diagnosed in 2001 with Ph+ CMLCP (low Sokalrisk).From 2001 to 2005 he was treated with Hydrea and Interferon with low dose Cytarabine. In 2005 he was switched to imatinib 400 mg/day. Due to cytogenetic resistance Imatinib dose was escalated to 600mg/day in 2006 and 800mg/day in 2007. No cytogenetic response was obtained. In 2009 patient was switched to nilotinib. Nilotinib therapy was discontinued in 2012 due to loss of complete hematologic response. Patient refuses unrelated donor allo HSCT. INFa 3000 U/3 times a day was given with prolonged stable complete hematologic response. Myeloid blasts crisis was diagnosed in Jan 2013. (The time patient was hospitalized to Almazov medical research center). Dasatinib 140mg/day was started without rapid blast reduction.The response lost in a few weeks, followed by 2 couses of FLAG regimen. Patient died in Dec 2013.
BCR-ABL KD mutations history: 2007- no mutations found; 2009- no mutations found, February 2013 -L248V mutation; July 2013 mutation analysis returns no result due to the poor sequence quality;T315I mutation revealed in samples obtained from October 2013.
BCR-ABL mutation analysis: Bcr-Abl analysis performed as described before (Branford et al., 2002). KD sequence was amplified from cDNA using seminested PCR, PCR product extracted from gel and sequenced in the both directions on ABI 3130 genetic analyzer. For confirmation we used genomic DNA extracted from stored peripheral blood samples. Primers specifically amplifying exon 4 and 5 of ABL used for genomic DNA analysis. Analysis of the sequences was done using UGENE software.
Results: We analyzed two sets of probes- one dated March 2011 and another dated October 2013. After performing semi-nested PCR on BCR-ABL KD sequence we found at least two bands in each of the samples. Sequencing of this bands revealed 81 bp deletion translated into absence of 27 aa position at 248-274. Genomic DNA analysis confirmed alternative splicing and appearance of the truncated form. We found SNP resulted in T315I mutation presented either in full or in short isoform of BCR-ABL in samples from October 2013. Also we confirmed L248V mutation presented in all analyzed samples.
Conclusion: SNP that cause L248V mutation in BCR-ABL transcript known to activate cryptic promotor in ABL exon4 with subsequent appearance of alternative splicing form described before (Gruber et al., 2006). Truncated form known to be kinase dead and have no influence on disease course (Shebenou et al., 2008). On the other hand it disturbs the Sanger sequence quality, masking the appearance of the additional mutations. Extensive attention requires in obtain proper sequence quality: use genomic DNA for Sanger sequencing or alternative sequencing methods, e.g. single-molecule long-read sequencing for revealing mutations in multiple splice isoforms.
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Disclosures
Lomaya: Novartis: Consultancy. Zaritskey:University of Heidelberg: Research Funding; Novartis: Consultancy.