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Recurrent internal tandem duplications of BCOR in clear cell sarcoma of the kidney.

Roy A, Kumar V, Zorman B, Fang E, Haines KM, Doddapaneni H, Hampton OA, White S, Bavle AA, Patel NR, Eldin KW, John Hicks M, Rakheja D, Leavey PJ, Skapek SX, Amatruda JF, Nuchtern JG, Chintagumpala MM, Wheeler DA, Plon SE, Sumazin P, Parsons DW - Nat Commun (2015)

Bottom Line: Identical ITD mutations are found in primary and relapsed tumour pairs but not in adjacent normal kidney or blood.Transcriptome analysis of ITD-positive CCSKs reveals enrichment for PRC2-regulated genes and similarity to undifferentiated sarcomas harbouring BCOR-CCNB3 fusions.The discovery of recurrent BCOR ITDs defines a major oncogenic event in this childhood sarcoma with significant implications for diagnostic and therapeutic approaches to this tumour.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology &Immunology, Baylor College of Medicine, Houston, Texas 77030, USA.

ABSTRACT
The X-linked BCL-6 co-repressor (BCOR) gene encodes a key constituent of a variant polycomb repressive complex (PRC) that is mutated or translocated in human cancers. Here we report on the identification of somatic internal tandem duplications (ITDs) clustering in the C terminus of BCOR in 23 of 27 (85%) pediatric clear cell sarcomas of the kidney (CCSK) from two independent cohorts. We profile CCSK tumours using a combination of whole-exome, transcriptome and targeted sequencing. Identical ITD mutations are found in primary and relapsed tumour pairs but not in adjacent normal kidney or blood. Mutant BCOR transcripts and proteins are markedly upregulated in ITD-positive tumours. Transcriptome analysis of ITD-positive CCSKs reveals enrichment for PRC2-regulated genes and similarity to undifferentiated sarcomas harbouring BCOR-CCNB3 fusions. The discovery of recurrent BCOR ITDs defines a major oncogenic event in this childhood sarcoma with significant implications for diagnostic and therapeutic approaches to this tumour.

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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.
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f1: 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.

Mentions: On closer inspection, WES tumour sequencing reads harbouring the variant BCOR allele were found to have adjacent soft clipping (Supplementary Fig. 1a). Notably, analysis of the aligned RNA-seq reads from all three tumours revealed similar soft-clipped subsequences (Supplementary Fig. 1b). Since soft clipping by mapping algorithms may be indicative of reads spanning genomic breakpoints of structural variations12, we analysed the clipped BCOR sequences using the Basic Local Alignment Search Tool13 and discovered in-frame ITDs within exon 15 of BCOR in all three cases (Table 1). Local realignment of discordant mate pairs showed a distinct focal increase in read coverage corresponding to the ITDs (Fig. 1a and Supplementary Fig. 2), which were subsequently confirmed by targeted PCR and sequencing (Fig. 1b,c).


Recurrent internal tandem duplications of BCOR in clear cell sarcoma of the kidney.

Roy A, Kumar V, Zorman B, Fang E, Haines KM, Doddapaneni H, Hampton OA, White S, Bavle AA, Patel NR, Eldin KW, John Hicks M, Rakheja D, Leavey PJ, Skapek SX, Amatruda JF, Nuchtern JG, Chintagumpala MM, Wheeler DA, Plon SE, Sumazin P, Parsons DW - Nat Commun (2015)

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.
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Related In: Results  -  Collection

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f1: 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.
Mentions: On closer inspection, WES tumour sequencing reads harbouring the variant BCOR allele were found to have adjacent soft clipping (Supplementary Fig. 1a). Notably, analysis of the aligned RNA-seq reads from all three tumours revealed similar soft-clipped subsequences (Supplementary Fig. 1b). Since soft clipping by mapping algorithms may be indicative of reads spanning genomic breakpoints of structural variations12, we analysed the clipped BCOR sequences using the Basic Local Alignment Search Tool13 and discovered in-frame ITDs within exon 15 of BCOR in all three cases (Table 1). Local realignment of discordant mate pairs showed a distinct focal increase in read coverage corresponding to the ITDs (Fig. 1a and Supplementary Fig. 2), which were subsequently confirmed by targeted PCR and sequencing (Fig. 1b,c).

Bottom Line: Identical ITD mutations are found in primary and relapsed tumour pairs but not in adjacent normal kidney or blood.Transcriptome analysis of ITD-positive CCSKs reveals enrichment for PRC2-regulated genes and similarity to undifferentiated sarcomas harbouring BCOR-CCNB3 fusions.The discovery of recurrent BCOR ITDs defines a major oncogenic event in this childhood sarcoma with significant implications for diagnostic and therapeutic approaches to this tumour.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology &Immunology, Baylor College of Medicine, Houston, Texas 77030, USA.

ABSTRACT
The X-linked BCL-6 co-repressor (BCOR) gene encodes a key constituent of a variant polycomb repressive complex (PRC) that is mutated or translocated in human cancers. Here we report on the identification of somatic internal tandem duplications (ITDs) clustering in the C terminus of BCOR in 23 of 27 (85%) pediatric clear cell sarcomas of the kidney (CCSK) from two independent cohorts. We profile CCSK tumours using a combination of whole-exome, transcriptome and targeted sequencing. Identical ITD mutations are found in primary and relapsed tumour pairs but not in adjacent normal kidney or blood. Mutant BCOR transcripts and proteins are markedly upregulated in ITD-positive tumours. Transcriptome analysis of ITD-positive CCSKs reveals enrichment for PRC2-regulated genes and similarity to undifferentiated sarcomas harbouring BCOR-CCNB3 fusions. The discovery of recurrent BCOR ITDs defines a major oncogenic event in this childhood sarcoma with significant implications for diagnostic and therapeutic approaches to this tumour.

Show MeSH
Related in: MedlinePlus