Figure 6.
Identification of an alternative CD19 splicing isoform (ex2part) associated with blinatumomab response. (A) Visualization of 3 CD19 isoforms: wild type (WT), ex2part, and ex2skip (Δex2). (B) Reverse transcription polymerase chain reaction of CD19 in KOPN75 cells showing 3 bands corresponding to CD19 WT, ex2part, and ex2skip (top). Sanger sequencing of CD19 ex2part in KOPN75 cells, confirming deletion of 130 nucleotides in exon 2 (bottom). (C) Consensus sequence assembled from Nanopore long-read RNA-seq identified the AceView isoform CD19 cAug10 with ex2part in 2 B-ALL patients. Numbers on top indicate coding sequence position; numbers on the right indicate RNA/protein position. IRE1-mediated splicing sequence CAGCCTGG indicated in red. (D) Full-length structure of CD19 WT and ex2part. Frame-shift partial deletion of exon 2 coding for amino acids Leu66 to Pro109 (ex2part) leads to a new start codon at Met152 of the canonical CD19. Translation of ex2part from the canonical start M1 results in a premature stop codon. (E) Levels of ex2part (E) and ex2skip (F) in pretreatment samples comparing responders (n = 13) and nonresponders (n = 15; left) and preblinatumomab (n = 13) compared with postblinatumomab relapse (n = 5; right). Median and interquartile range are displayed. P value determined using Mann-Whitney test. mRNA, messenger RNA; ORF, open reading frame.

Identification of an alternative CD19 splicing isoform (ex2part) associated with blinatumomab response. (A) Visualization of 3 CD19 isoforms: wild type (WT), ex2part, and ex2skip (Δex2). (B) Reverse transcription polymerase chain reaction of CD19 in KOPN75 cells showing 3 bands corresponding to CD19 WT, ex2part, and ex2skip (top). Sanger sequencing of CD19 ex2part in KOPN75 cells, confirming deletion of 130 nucleotides in exon 2 (bottom). (C) Consensus sequence assembled from Nanopore long-read RNA-seq identified the AceView isoform CD19 cAug10 with ex2part in 2 B-ALL patients. Numbers on top indicate coding sequence position; numbers on the right indicate RNA/protein position. IRE1-mediated splicing sequence CAGCCTGG indicated in red. (D) Full-length structure of CD19 WT and ex2part. Frame-shift partial deletion of exon 2 coding for amino acids Leu66 to Pro109 (ex2part) leads to a new start codon at Met152 of the canonical CD19. Translation of ex2part from the canonical start M1 results in a premature stop codon. (E) Levels of ex2part (E) and ex2skip (F) in pretreatment samples comparing responders (n = 13) and nonresponders (n = 15; left) and preblinatumomab (n = 13) compared with postblinatumomab relapse (n = 5; right). Median and interquartile range are displayed. P value determined using Mann-Whitney test. mRNA, messenger RNA; ORF, open reading frame.

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