Identification of Novel MET Exon 14 Skipping Variants in Non-Small Cell Lung Cancer Patients: A Prototype Workflow Involving in Silico Prediction and RT-PCR

• Published in: Cancers 2022-10, Vol.14 (19), p.4814
• Main Authors: Das, Riku, Jakubowski, Maureen A, Spildener, Jessica, Cheng, Yu-Wei
• Format: Article
• Language: English
• Subjects: Alternative splicing; Binding sites; Cancer therapies; Cellular biology; Cytology; Data analysis; Diagnosis; Exon (Molecular genetics); FDA approval; Gene amplification; Gene deletion; Genetic aspects; Genetic markers; Genetic screening; Genomes; Health aspects; Kinases; Lung cancer; Lung cancer, Non-small cell; Mutation; Mutation (Biology); Next-generation sequencing; Non-small cell lung carcinoma; Patients; Polymerase chain reaction; Predictions; Reverse transcription; Ribonucleic acid; RNA; Sequence analysis; Small cell lung carcinoma; Splicing
• ISSN: 2072-6694; 2072-6694
• DOI: 10.3390/cancers14194814

Background and aims: The MET exon 14 skipping (METex14) is an oncogenic driver mutation that provides a therapeutic opportunity in non-small cell lung cancer (NSCLCs) patients. This event often results from sequence changes at the MET canonical splicing sites. We characterize two novel non-canonical splicing site variants of MET that produce METex14. Materials and Methods: Two variants were identified in three advanced-stage NSCLC patients in a next-generation sequencing panel. The potential impact on splicing was predicted using in silico tools. METex14 mutation was confirmed using reverse transcription (RT)-PCR and a Sanger sequencing analysis on RNA extracted from stained cytology smears. Results: The interrogated MET (RefSeq ID NM_000245.3) variants include a single nucleotide substitution, c.3028+3A>T, in intron 14 and a deletion mutation, c.3012_3028del, in exon 14. The in silico prediction analysis exhibited reduced splicing strength in both variants compared with the MET normal transcript. The RT-PCR and subsequent Sanger sequencing analyses confirmed METex14 skipping in all three patients carrying these variants. Conclusion: This study reveals two non-canonical MET splice variants that cause exon 14 skipping, concurrently also proposes a clinical workflow for the classification of such non-canonical splicing site variants detected by routine DNA-based NGS test. It shows the usefulness of in silico prediction to identify potential METex14 driver mutation and exemplifies the opportunity of routine cytology slides for RNA-based testing.