Balance potentially introduced by contaminating CLL cells, we tested the non-B cell fraction of the enriched mononuclear cells (referred to as “flow-through”). CLL negative fractions of PBMC were generated by CD19 depletion in 11 informative CLL patients. FACS, detecting contaminating CLL cell populations less than 2 , assured the efficacy of CD19 depletion in these CLL patient samples. A similarly pronounced widespread distribution of allelic imbalances could be observed (Figure S3), indicating that DAPK1 ASE occurs in non-malignant cell populations and thus it is likely to be a germline feature. By comparing the baseline clinical characteristics of the 14 imbalanced to the 30 most balanced CLL cases, we could not observe any statistically significant differences between the two groups. However, the age at diagnosis showed a clear trend towards earlier onset in the ASE CAL-120 site positive group (median age = 53.0, range = 40?1 years) compared to the balanced cases (median age = 62.5, range = 41?6 years, p = 0.044). No differences in survival endpoints (overall survival, time to treatment failure) or other relevant disease characteristics known to predict prognosis (IGHV mutational status, cytogenetics) were detected.An epigenetic cause of DAPK1 ASE in CLLASE could potentially be explained by different mechanisms. Sequence analysis of all DAPK1 exons in 96 CLL patient samples showed the presence of previously reported SNPs in exons 3, 4, 16 and 26. However, no mutations in the coding sequence that may result in nonsense mediated RNA decay were revealed. No modifications were identified in the 39 `UTR of DAPK1 that might interfere with (or create new) miRNA binding sites. We alsoAllele-Specific Expression of DAPK1 in CLLFigure 2. DAPK1 allele-specific expression (ASE) in CLL patients. (A) 120 CLL cases and 63 controls were analyzed for DAPK1 ASE using the informative SNP rs1056719 (G/A) as outlined previously. Allelic ratios (in relation to the G allele) of DAPK1 mRNA from peripheral blood mononuclear cells (PBMCs) were measured with the outlined SNuPE/MALDI-TOF-based method. Dashed lines mark statistically (Youden index outlier method) determined thresholds to identify ASE positive outliers contributing to the significant variability of CLL compared to healthy controls. The centre of these thresholds (0.29 and 0.54, dashed lines) is the estimated average (0.4) of the 15857111 CLL sample group. (B) Scheme of the DAPK1 promoter SR-3029 manufacturer region with grey boxes representing the first 2 exons of DAPK1. Nucleotide positions are given relative to DAPK1 transcriptional start site (TSS). Dashed lines represent positions of the investigated regions/amplicons. (C) Quantitative DNA methylation analysis in the amplicons A, C and D (as described in figure 4) for the seven most imbalanced and seven most balanced CLL patients with regard to DAPK1 mRNA expression. Scatter plots represent mean amplicon methylation levels. Significance was assessed by non-parametric Mann-Whitney-U test (* indicates p,0.01). doi:10.1371/journal.pone.0055261.ginvestigated a 27 kb to +2 kb region around the DAPK1 transcriptional start site for a haplotype associated with allelic expression imbalances. This region constitutes a genomic block with high genetic linkage disequilibrium. Fifteen SNPs were genotyped in ASE positive and negative CLL samples, however, no segregation of a genotype/haplotype with the presence of ASE could be detected. To search for evidence that epigenetic alterations (e.g. prom.Balance potentially introduced by contaminating CLL cells, we tested the non-B cell fraction of the enriched mononuclear cells (referred to as “flow-through”). CLL negative fractions of PBMC were generated by CD19 depletion in 11 informative CLL patients. FACS, detecting contaminating CLL cell populations less than 2 , assured the efficacy of CD19 depletion in these CLL patient samples. A similarly pronounced widespread distribution of allelic imbalances could be observed (Figure S3), indicating that DAPK1 ASE occurs in non-malignant cell populations and thus it is likely to be a germline feature. By comparing the baseline clinical characteristics of the 14 imbalanced to the 30 most balanced CLL cases, we could not observe any statistically significant differences between the two groups. However, the age at diagnosis showed a clear trend towards earlier onset in the ASE positive group (median age = 53.0, range = 40?1 years) compared to the balanced cases (median age = 62.5, range = 41?6 years, p = 0.044). No differences in survival endpoints (overall survival, time to treatment failure) or other relevant disease characteristics known to predict prognosis (IGHV mutational status, cytogenetics) were detected.An epigenetic cause of DAPK1 ASE in CLLASE could potentially be explained by different mechanisms. Sequence analysis of all DAPK1 exons in 96 CLL patient samples showed the presence of previously reported SNPs in exons 3, 4, 16 and 26. However, no mutations in the coding sequence that may result in nonsense mediated RNA decay were revealed. No modifications were identified in the 39 `UTR of DAPK1 that might interfere with (or create new) miRNA binding sites. We alsoAllele-Specific Expression of DAPK1 in CLLFigure 2. DAPK1 allele-specific expression (ASE) in CLL patients. (A) 120 CLL cases and 63 controls were analyzed for DAPK1 ASE using the informative SNP rs1056719 (G/A) as outlined previously. Allelic ratios (in relation to the G allele) of DAPK1 mRNA from peripheral blood mononuclear cells (PBMCs) were measured with the outlined SNuPE/MALDI-TOF-based method. Dashed lines mark statistically (Youden index outlier method) determined thresholds to identify ASE positive outliers contributing to the significant variability of CLL compared to healthy controls. The centre of these thresholds (0.29 and 0.54, dashed lines) is the estimated average (0.4) of the 15857111 CLL sample group. (B) Scheme of the DAPK1 promoter region with grey boxes representing the first 2 exons of DAPK1. Nucleotide positions are given relative to DAPK1 transcriptional start site (TSS). Dashed lines represent positions of the investigated regions/amplicons. (C) Quantitative DNA methylation analysis in the amplicons A, C and D (as described in figure 4) for the seven most imbalanced and seven most balanced CLL patients with regard to DAPK1 mRNA expression. Scatter plots represent mean amplicon methylation levels. Significance was assessed by non-parametric Mann-Whitney-U test (* indicates p,0.01). doi:10.1371/journal.pone.0055261.ginvestigated a 27 kb to +2 kb region around the DAPK1 transcriptional start site for a haplotype associated with allelic expression imbalances. This region constitutes a genomic block with high genetic linkage disequilibrium. Fifteen SNPs were genotyped in ASE positive and negative CLL samples, however, no segregation of a genotype/haplotype with the presence of ASE could be detected. To search for evidence that epigenetic alterations (e.g. prom.
