Figure 3. Secondary splice site mutations drive alternative BRCA1 isoform expression.

(A) Schematic in section i) shows the BRCA1 exon 11q donor splice site and its interaction with the exon 12 acceptor splice site under wildtype conditions with no exon skipping. When the donor site is disrupted by a mutation (section ii), like in PDX #049 and COV362, the exon 11p donor site can be used preferentially, thus leading to 11q region skipping. In PDX #56PP the exon 11 acceptor site is disrupted by a large deletion, thus the next acceptor site for exon 12 may be used instead, leading to full exon 11 skipping (section iii). (B) This schematic shows the BRCA1 mini-gene design, and splicing outcomes predicted for each secondary splice site mutation found in PDX and cell line models. (C) Splicing predictions for each secondary splice-site mutation modelled by the mini-gene were confirmed by immunoblotting for the HA tag. The PDX #56PP deletion was confirmed to drive high Δ11 and potentially also isoform Δ(9,10,11q) expression. COV362 and PDX #049 secondary splice site mutations were confirmed to drive high Δ11q relative to the wild-type (WT) BRCA1 control and the primary deleterious BRCA1 mutation found in PDX #206. (D) Archival material (pre-PARPi) for patient #049 was screened for the secondary splice site mutation found in PDX #049. While the ascites from which the PDX was derived harboured the splice site mutation, the archival sample did not. (E) Archival material from the original debulking surgery (1C), or in a sample collected after 1^st line platinum therapy (3A and a second DNA extraction 3A_21), for PDX #56PP was tested for the secondary splice site deletion found in the PDX using highly sensitive droplet digital PCR. While no deletion was detected in archival samples, poor droplet amplification in these samples limits interpretation. T2 = transplant/passage 2 PDX aliquot.