Alternative pre-mRNA splicing increases transcriptome plasticity by forming naturally-occurring alternative splicing variants (ASVs). Alterations of splicing processes, caused by DNA mutations, result in aberrant splicing and the formation of aberrant mRNA isoforms. Analyses of hereditary cancer predisposition genes reveal many DNA variants with unknown clinical significance (VUS) that potentially affect pre-mRNA splicing. Therefore, a comprehensive description of ASVs is an essential prerequisite for the interpretation of germline VUS in high-risk individuals. To identify ASVs in a gene of interest, we have proposed an approach based on multiplex PCR (mPCR) amplification of all theoretically possible exon-exon junctions and subsequent characterization of size-selected and pooled mPCR products by next-generation sequencing (NGS). The efficiency of this method is illustrated by a comprehensive analysis of BRCA1 ASVs in human leukocytes, normal mammary, and adipose tissues and stable cell lines. We revealed 94 BRCA1 ASVs, including 29 variants present in all tested samples. While differences in the qualitative expression of BRCA1 ASVs among the analyzed human tissues were minor, larger differences were detected between tissue and cell line samples. Compared with other ASV analysis methods, this approach represents a highly sensitive and rapid alternative for the identification of ASVs in any gene of interest.

Department of Obstetrics and Gynecology 1st Faculty of Medicine Charles University and General University Hospital Prague Prague 120 00 Czech Republic

Department of Oncology 1st Faculty of Medicine Charles University and General University Hospital Prague Prague 120 00 Czech Republic

Department of Plastic Surgery 1st Faculty of Medicine Charles University and Na Bulovce Hospital Prague 180 81 Czech Republic

Institute of Biochemistry and Experimental Oncology 1st Faculty of Medicine Charles University Prague 12853 Czech Republic

Institute of Biology and Medical Genetics 1st Faculty of Medicine Charles University and General University Hospital Prague Prague 120 00 Czech Republic

Institute of Inherited Metabolic Disorders 1st Faculty of Medicine Charles University and General University Hospital Prague Prague 120 00 Czech Republic

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$a Multiplex PCR and NGS-based identification of mRNA splicing variants: Analysis of BRCA1 splicing pattern as a model / $c J. Hojny, P. Zemankova, F. Lhota, J. Sevcik, V. Stranecky, H. Hartmannova, K. Hodanova, O. Mestak, D. Pavlista, M. Janatova, J. Soukupova, M. Vocka, Z. Kleibl, P. Kleiblova,

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$a Alternative pre-mRNA splicing increases transcriptome plasticity by forming naturally-occurring alternative splicing variants (ASVs). Alterations of splicing processes, caused by DNA mutations, result in aberrant splicing and the formation of aberrant mRNA isoforms. Analyses of hereditary cancer predisposition genes reveal many DNA variants with unknown clinical significance (VUS) that potentially affect pre-mRNA splicing. Therefore, a comprehensive description of ASVs is an essential prerequisite for the interpretation of germline VUS in high-risk individuals. To identify ASVs in a gene of interest, we have proposed an approach based on multiplex PCR (mPCR) amplification of all theoretically possible exon-exon junctions and subsequent characterization of size-selected and pooled mPCR products by next-generation sequencing (NGS). The efficiency of this method is illustrated by a comprehensive analysis of BRCA1 ASVs in human leukocytes, normal mammary, and adipose tissues and stable cell lines. We revealed 94 BRCA1 ASVs, including 29 variants present in all tested samples. While differences in the qualitative expression of BRCA1 ASVs among the analyzed human tissues were minor, larger differences were detected between tissue and cell line samples. Compared with other ASV analysis methods, this approach represents a highly sensitive and rapid alternative for the identification of ASVs in any gene of interest.

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$a Zemankova, Petra $u Institute of Biochemistry and Experimental Oncology, First Faculty of Medicine, Charles University, Prague 12853, Czech Republic.

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$a Sevcik, Jan $u Institute of Biochemistry and Experimental Oncology, First Faculty of Medicine, Charles University, Prague 12853, Czech Republic.

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$a Stranecky, Viktor $u Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague 120 00, Czech Republic.

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$a Hartmannova, Hana $u Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague 120 00, Czech Republic.

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$a Hodanova, Katerina $u Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague 120 00, Czech Republic.

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$a Mestak, Ondrej $u Department of Plastic Surgery, First Faculty of Medicine, Charles University and Na Bulovce Hospital, Prague 180 81, Czech Republic.

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$a Pavlista, David $u Department of Obstetrics and Gynecology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague 120 00, Czech Republic.

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$a Janatova, Marketa $u Institute of Biochemistry and Experimental Oncology, First Faculty of Medicine, Charles University, Prague 12853, Czech Republic.

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$a Soukupova, Jana $u Institute of Biochemistry and Experimental Oncology, First Faculty of Medicine, Charles University, Prague 12853, Czech Republic.

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$a Vocka, Michal $u Department of Oncology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague 120 00, Czech Republic.

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$a Kleibl, Zdenek $u Institute of Biochemistry and Experimental Oncology, First Faculty of Medicine, Charles University, Prague 12853, Czech Republic.

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$a Kleiblova, Petra $u Institute of Biochemistry and Experimental Oncology, First Faculty of Medicine, Charles University, Prague 12853, Czech Republic; Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague 120 00, Czech Republic. Electronic address: petra.kleiblova@lf1.cuni.cz.

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