Figure 2.
Cooperating genomic events define gene expression subclusters, including alternative HBS1L isoform expression in delHBS1L and del7 as novel candidate. (A) Distribution of recurrent copy number variants (CNVs) in the 4 BCR::ABL1-positive ALL subclusters. CNVs were assessed in samples with subcluster allocation (GMALL: ground truth; MLL: predictions, excluding n = 14 samples that remained "unclassified" by machine learning classifier for the 2- and/or the 4-cluster definition) by whole-genome sequencing (WGS) (n = 47) or single-nucleotide polymorphism (SNP) array (n = 102) and validated by fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR), and/or multiplex ligation-dependent probe amplification (MLPA). The identified recurrent HBS1L deletion harbored the same breakpoints in all samples as identified by WGS (chr6:135,044,863-135,116,862; GRCh38hg38), including the HBS1L promoter and exon 1 to 2. Bars represent the percentage of BCR::ABL1-positive cases with a given alteration within each category. Associations between delHBS1L vs del7 vs IKZF1 vs CDKN2A/PAX5 were assessed by χ2 or Fisher exact test (P values below the significance level of 0.05 are depicted in bold). For detailed statistic please refer to supplemental Figure 5 and supplemental Appendix. (B) Subcluster-specific patterns of genomic aberrations were validated in the PMCC cohort (n = 49) using subcluster allocations obtained from a machine learning classifier trained on the GMALL cohort and the published14 genomic aberration profile of these samples. (C) Hierarchical clustering was performed using HBS1L alternative transcription start side expression (TSS; chr6: 135,040,344-135,040,447), HBS1L exon use, and HBS1L total gene expression in 113 GMALL samples. In addition, the average expression on HBS1L exons 1 to 3 is shown. (D) Direct long-read RNA-sequencing reads of HBS1L region between exons 1 and 4 are shown for 1 lymphoid and 1 multilineage BCR::ABL1-positive sample. The predicted alternative promoter in the intronic region between exon 3 and 4 is depicted in red. The orange bar shows the identified genomic deletion in HBS1L. A more detailed overview of the alternative HBS1L transcript and confirmation of the alternative TSS by single-cell ATAC-Seq is shown in supplemental Figures 7 and 8.)

Cooperating genomic events define gene expression subclusters, including alternative HBS1L isoform expression in delHBS1L and del7 as novel candidate. (A) Distribution of recurrent copy number variants (CNVs) in the 4 BCR::ABL1-positive ALL subclusters. CNVs were assessed in samples with subcluster allocation (GMALL: ground truth; MLL: predictions, excluding n = 14 samples that remained "unclassified" by machine learning classifier for the 2- and/or the 4-cluster definition) by whole-genome sequencing (WGS) (n = 47) or single-nucleotide polymorphism (SNP) array (n = 102) and validated by fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR), and/or multiplex ligation-dependent probe amplification (MLPA). The identified recurrent HBS1L deletion harbored the same breakpoints in all samples as identified by WGS (chr6:135,044,863-135,116,862; GRCh38hg38), including the HBS1L promoter and exon 1 to 2. Bars represent the percentage of BCR::ABL1-positive cases with a given alteration within each category. Associations between delHBS1L vs del7 vs IKZF1 vs CDKN2A/PAX5 were assessed by χ2 or Fisher exact test (P values below the significance level of 0.05 are depicted in bold). For detailed statistic please refer to supplemental Figure 5 and supplemental Appendix. (B) Subcluster-specific patterns of genomic aberrations were validated in the PMCC cohort (n = 49) using subcluster allocations obtained from a machine learning classifier trained on the GMALL cohort and the published14 genomic aberration profile of these samples. (C) Hierarchical clustering was performed using HBS1L alternative transcription start side expression (TSS; chr6: 135,040,344-135,040,447), HBS1L exon use, and HBS1L total gene expression in 113 GMALL samples. In addition, the average expression on HBS1L exons 1 to 3 is shown. (D) Direct long-read RNA-sequencing reads of HBS1L region between exons 1 and 4 are shown for 1 lymphoid and 1 multilineage BCR::ABL1-positive sample. The predicted alternative promoter in the intronic region between exon 3 and 4 is depicted in red. The orange bar shows the identified genomic deletion in HBS1L. A more detailed overview of the alternative HBS1L transcript and confirmation of the alternative TSS by single-cell ATAC-Seq is shown in supplemental Figures 7 and 8.)

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