Figure 1: Recurrent somatic ITDs in the BCOR gene in CCSKs.

(a) View of aligned whole-transcriptome sequencing reads from a single ITD-positive CCSK (347T) demonstrating a marked focal increase in read coverage corresponding to the ITD in exon 15 of BCOR on Xp11.4. Only unpaired reads of discordant mate pairs were used for local realignment. A representation of the ITD within the BCOR gene is shown beneath. The parental segment (P) that is duplicated is depicted in green and the tandem duplicated segment (ITD) is shown in red. (b) Targeted PCR and gel electrophoresis of BCOR exon 15 in samples from four representative male and two female subjects showed the expected wild-type products (288 bp) in the peripheral blood (C) and adjacent normal kidney (N) tissues, and larger products corresponding to the ITDs (87–114 bp) in the primary CCSK tumours (T) and relapsed metastatic tumours (R). Nearly undetectable levels of the wild-type products were observed in tumour samples from males; in females, both ITD-positive and wild-type products were evident. (c) Sanger sequence trace from case 347T showing the immediate sequence context surrounding the proximal genomic breakpoint in BCOR exon 15 (hg19 coordinates, negative strand). The wild-type genomic sequence around the breakpoint including the termination codon and 3′-UTR are shown above and the parental and duplicated segments of the ITD are below. The proximal breakpoint at the second base of the stop codon (TGA) alters it to a TTA (leucine). (d) Schematic of predicted BCOR protein sequences from ITD-positive CCSKs demonstrating the clustering of all ITDs within the C-terminal PUFD domain. ITD types I–V were numbered based on genomic breakpoints and ITD sequence (Table 1). The BCOR wild-type protein sequence (amino acids (aa) 1,701–1,755) is shown on top with the predicted protein sequence of each ITD-positive case below. Parental segments that have been duplicated are shown in green and the ITDs in red. Novel junctional amino acids (bold black font, underlined) were introduced by the ITDs in cases 385T and 504T. A stretch of 14 residues (aa 1,724–1,737) is common to every ITD type. ANK, ankyrin repeats; BBD, BCL6-binding domain.