RNA- Sequencing data supports the existence of novel VEGFA splicing events but not of VEGFAxxxb isoforms

Vascular endothelial growth factor (VEGFA), a pivotal regulator of angiogenesis and valuable therapeutic target, is characterised by alternative splicing which generates three principal isoforms, VEGFA(121), VEGFA(165) and VEGFA(189). A second set of anti-angiogenic isoforms termed VEGFA(xxx)b that utilise an alternative splice site in the final exon have been widely reported, with mRNA detection based principally upon RT-PCR assays. We sought confirmation of the existence of the VEGFA(xxx)b isoforms within the abundant RNA sequencing data available publicly. Whilst sequences derived specifically from each of the canonical VEGFA isoforms were present in many tissues, there were no sequences derived from VEGFA(xxx)b isoforms. Sequencing of approximately 50,000 RT-PCR products spanning the exon 7-8 junction in 10 tissues did not identify any VEGFA(xxx)b transcripts. The absence or extremely low expression of these transcripts in vivo indicates that VEGFA(xxx)b isoforms are unlikely to play a role in normal physiology. Our analyses also revealed multiple novel splicing events supported by more reads than previously reported for VEGFA(145) and VEGFA(148) isoforms, including three from novel first exons consistent with existing transcription start site data. These novel VEGFA isoforms may play significant roles in specific cell types.