Abstract A183: Identification of an exon 31-deletion variant of Notch1 receptor with frequent expression in head and neck cancer.
Authors: Psyrri Amanda , Pectasides Eirini , Avgeris M , Klinakis Apostolos , Scorilas Andreas , Sasaki Clarence , Fountzilas George , Rampias Theodoros 
Published: August 2014

Abstract
Introduction: Recently, whole genome sequencing studies revealed that loss of function somatic mutations in NOTCH1 receptor are present in more than 15% of human head and neck cancers making NOTCH1 signalling an attractive target for the development of new, highly specific antitumor drugs. Here, we present a novel Notch 1 receptor variant containing an in frame deletion of 231 nucleotides (named as del31 Notch) of exon 31.
Materials and Methods: Total RNA from 64 frozen head and neck and 72 bladder cancer and matched normal tissue were used for cDNA synthesis by conventional reverse transcription using random primers. In a next step, the fragments corresponding to the full size Notch1 receptor were PCR amplified and subjected to Sanger sequencing analysis. We analysed the sequencing data, focusing on truncation and exonic deletion events in the Notch 1 receptor.
Results: During PCR amplification of the cDNA fragment corresponding to exon 31 of Notch 1 receptor in our samples, we identified an unexpected PCR product that was slightly smaller in size than wild type exon. Sequencing of this product revealed a Notch1 truncated transcript that lacks a part of exon 31 sequence, harbouring a 231 nt in frame deletion. Further characterization of the truncated receptor revealed that its intracellular domain contains a shorter ankyrin domain (ANK) and that its transactivation efficiency is less than 65% compared to the wild type receptor. Sequencing analysis of genomic DNA in our samples revealed that this exonic deletion mutation was not present in genomic DNA, suggesting that the truncated variant is possibly generated by an abnormal splicing event. In support to this proposed mechanism, the 5’ end of the deleted sequencing is located near to 3’ acceptor slice site of the intron between exons 30 and 31. To identify the incidence of Notch1 del31 variant in cancer specimens, we designed a Notch1 del31 variant-specific PCR assay. Variant specific polymerase chain reaction showed a high incidence of Notch1 del31 variant expression in head and neck cancer (8/64), while the incidence of this variant was rare in bladder cancer (1/72). We did not observe expression of Notch1 del31 variant in tissue adjacent to tumors or in normal tissues.
Discussion: We present a novel variant of Notch1 receptor which exhibits lower transactivation activity compared to wild type receptor. Loss of function mutations of Notch1 are associated with head and neck carcinogenesis.Therefore, Notch1 del 31 truncated variant may promote carcinogenesis by suppressing the Notch signalling in head and neck epithelium.
Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A183.
Citation Format: Amanda Psyrri, Eirini Pectasides, M Avgeris, Apostolos Klinakis, Andreas Scorilas, Clarence Sasaki, George Fountzilas, Theodoros Rampias. Identification of an exon 31-deletion variant of Notch1 receptor with frequent expression in head and neck cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A183.