Novel Assay for the Identification of NOTCH1 PEST Domain Mutations Using Fragment Analysis and Allele Specific PCR
Authors: Campregher Paulo Vidal , Petroni Roberta Cardoso , Muto Nair , Santana Rubia , Sitnik Roberta , Ramos Oziris P S , Bacal Nydia , Bub Carolina B , Santos Fabio P S , Guilherme Perini , Ricardo Helman , Souza Clarissa , de Assis Reijane Alves , Oliveira Gislaine Borba , Metze Irene L , Hamerschlak Nelson 
Published: October 2019

Abstract
Abstract 5127
NOTCH1 is a proto-oncogene with activating mutations described in a variety of malignancies, including acute lymphoblastic leukemia (ALL), mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL). While the prognostic significance of NOTCH1 mutations remains controversial in ALL, recent data suggest that NOTCH1 PEST domain mutations are associated with adverse prognosis in patients with CLL. NOTCH1 mutations are found in around 8% of CLL patients at diagnosis and more than 30% of patients with advanced disease. Since this disease has a heterogeneous clinical course and few prognostic markers, we aimed at designing a fast, cost effective and robust assay to detect NOTCH1 PEST domain mutations in patients with CLL for the clinical laboratory. While 92% of the mutations in NOTCH1 PEST domain found in CLL are insertions or deletions, only 8% are represented by point mutations. Therefore we decided to use a fragment analysis approach in our assay. Given that a single mutation (c. 7544_7545delCT), represents roughly 75% of all PEST domain mutations in CLL we designed a test that can, at the same time, detect the presence of this mutation specifically and also any insertion or deletion in exon 34.
We designed a PCR reaction using one FAM-labeled forward primer anchored at codon 2407 and two reverse primers. One specific for the c. 7544_7545delCT mutation anchored at codon 2414 yielding a product of 356 base pairs (bp) and one anchored at codon 2425, yielding a product of 391 bp, comprising the hot spot for mutations in the NOTCH1 PEST domain. Primers were designed with Primer3 software (http://frodo.wi.mit.edu/) and the specificity of the reaction evaluated using the tool “PCR in silico” (http://genome.ucsc.edu/cgi-bin/hgPcr?command=start). The test yields three possible outputs: A single 391 bp peak: wild type samplesThree peaks (391 bp, 389 bp and 356 bp): heterozygous for c. 7544_7545delCTTwo peaks (391 bp and another bigger or smaller, depending on the size of insertion/deletion): another insertion or deletion, but not c. 7544_7545delCT.
We have studied 46 de-identified blood samples from patients with CLL, in several diverse stages, using our assay. In 40 patients, there was no NOTCH1 mutation detected. Six patients had a pattern compatible with c. 7544_7545delCT NOTCH1 mutation (see figure 1), and no patients presented with another mutation. Overall the frequency of NOTCH1 mutations in our series was 13 %. Selected mutated samples were confirmed through amplicon sequencing.
In conclusion, we have designed a robust, fast and cost effective assay for routine identification of NOTCH1 PEST domain mutations using fragment analysis and allele specific pcr that is suitable for implementation in the clinical setting for CLL patients evaluation. We will continue testing more CLL patients in order to identify another, rarer, NOTCH1 mutations.
Figure 1. Assay Results for NOTCH1 PEST Domain Mutations A – Wild Type NOTCH1 revealed by the presence of a single 391 bp peak. B – Presence of heterozygous c. 7544_7545delCT mutation evidenced by the presence of a 356 bp peak, corresponding to the allele specific pcr peak; and a double peak at 391 bp and 389 bp positions, corresponding to the wild type product (391 bp) and to the mutated product (389 bp) detected with the wild type primers. Figure 1. Assay Results for NOTCH1 PEST Domain Mutations . / A – Wild Type NOTCH1 revealed by the presence of a single 391 bp peak. . / B – Presence of heterozygous c. 7544_7545delCT mutation evidenced by the presence of a 356 bp peak, corresponding to the allele specific pcr peak; and a double peak at 391 bp and 389 bp positions, corresponding to the wild type product (391 bp) and to the mutated product (389 bp) detected with the wild type primers.

Disclosures:
No relevant conflicts of interest to declare.