NOTCH2 missplicing can occur in relation to apoptosis

To the editor:

In a recent study in Blood, Adamia et al have identified from an analysis on RNA sequencing and exon array data, that NOTCH2 and FLT3 gene missplicings are common events and potential targets in acute myeloid leukemia (AML).¹  Among the splice variants, NOTCH2-Va resulting from a complete deletion of exon 12 (111 bp) was of particular interest because it was detected at a very high frequency (73%) in AML and its high expression was associated with poor outcome in patients with an intermediate-risk cytogenetics.

Incidentally, we found that this splice variant can be observed in normal hematopoietic cells undergoing apoptosis. To further investigate this phenomenon, we obtained 8 bottles of blood specimens each from 3 healthy individuals and stored samples in a dark chamber at room temperature. RNA was extracted immediately after venipuncture and also sequentially after scheduled storage times: 1, 3, 6, and 12 hours and 1, 3, and 7 days. Fluorescence polymerase chain reaction (PCR) and capillary electrophoresis using primers to detect NOTCH2-Va and full-length wild-type (WT) transcript (NOTCH2-FL)¹  showed a gradual increase of NOTCH2-Va and a decrease of NOTCH2-FL during specimen aging (Figure 1A-B). Gel extraction and sequencing analysis for the abnormal PCR band confirmed skipping of the entire exon 12 of NOTCH2 (Figure 1C).

In line with our observations, another study also identified aberrant splicing of NOTCH2 in incubated normal blood cells by RNA sequencing,²  suggesting that the presence of NOTCH-Va may reflect the illegitimate splicing typically observed in specimen aging.³  The aberrant splicing is suggested to be widespread across the genome and associated with apoptosis pathways.^4,5  However, Adamia et al¹  have also demonstrated some functional consequences of the NOTCH2 splicing variants to induce downregulation of its target genes, such as HES1 and DTX1. Considering that deregulation of apoptosis is a key mechanism in the pathogenesis of AML,⁶  there might be some commonality between aging-related and leukemia-related missplicing events. And of special note, NOTCH2 is proven to have important roles on the regulation of apoptosis.^7,8  The original investigators have suggested NOTCH2 variants as attractive targets for novel therapeutics, and we may say that the targeting strategy should be considered in the context of cell apoptosis. For clinical diagnostics, it is strongly recommended that these variants be tested using fresh specimens and to interpret the results with an understanding of the dynamics of splicing during cellular aging.