Rrently we do not know if and how germline epigenetic alterations could affect DAPK1 expression and thereby might contribute to the Lixisenatide biological activity predisposing mechanism. Additional molecular mechanisms causing ASE could be nonsense-mediated mRNA decay, due to a mutation in the target gene, which seems to be a rare event. Another possible mechanism could be the modulation of miRNA binding due to sequence variations or sequence alterations affecting the promoter activity of cancer genes. High throughput genome analysis hasAllele-Specific Expression of DAPK1 in CLLuncovered copy number variations occurring throughout genomes 1531364 of healthy individuals. These variations could result in allelic imbalances of gene expression. Similarly, differentially methylated regions other than at imprinted regions can have similar effects on gene expression. The extent to which these mechanisms participate in ASE and cancer predisposition needs to be determined in future studies. Furthermore, prospective trials are needed to confirm these findings and to extend a predisposing role of DAPK1 ASE to non-malignant CLL precursor states like monoclonal Bcell lymphocytosis.ligated PCR products of the DAPK1 cDNA. rs1056719 indicates the polymorphic site, the arrows represent the cloning primers and indicate the sequencing direction. (B) Chromatograms representing the genomic region around the polymorphic site rs1056719 in Granta-519 cells. The upper panel displays the cDNA, the lower panel displays the genomic DNA as balancing control. (C) Chromatograms representing the polymorphic site rs3818584 in EHEB cells according to figure 3B. (TIF)Figure S6 Re-balancing of DAPK1 mRNA expression inSupporting InformationFigure S1 Accurate quantification of DAPK1 ASE investigating all four common exonic SNPs. Standard curves for plasmid based standards displaying allelic ratios from 1:50 to 50:1 and correlation with idealized ratios. (A) SNP rs3118863, DAPK1 exon 26, (B) SNP rs3818584, DAPK1 exon 16, (C) SNP rs36207428, DAPK1 exon 3. (D) SNP rs1056719, DAPK1 exon 26. (TIF) Figure S2 Detection sensitivity for quantitative genotyping of rs1056719. (A) Standard curves for plasmid molecular standard (A: 30,000 template plasmid copies, B: 300 template plasmid copies) and comparison with ideal linear correlation. Standard deviations are given for 4 replicate measurements. (TIF) Figure S3 DAPK1 ASE in CD19 depleted PBMC samples from CLL patients. (A) 120 CLL cases, 11 CD19 depleted (contaminating CD19+ population less than 2 ) 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 were measured with the outlined SNuPE/MALDI-TOF-based 3PO web method. (TIF) Figure S4 MEC-1 cells are fully methylated at the CpG island of the DAPK1 59 region. (A) Scheme of the DAPK1 promoter region and the associated CpG island. Grey boxes display the first 2 exons of DAPK1. Nucleotide positions are given relative to the DAPK1 transcriptional start site. Dashed lines represent positions of investigated regions/amplicons. (B) Quantitative DNA methylation analysis of the DAPK1 gene 59 region (amplicons A ) in untreated, control (PBS)-treated and 5-aza-29deoxycytidine (DAC)-treated MEC-1 cells was performed using the MassCleave method. Bars represent quantitative DNA methylation values ( ) at single CpG units. (TIF) Figure S5 Detection of DAPK1 ASE by conventionalGranta-519 cells upon inhibition of DNA methyltransferas.Rrently we do not know if and how germline epigenetic alterations could affect DAPK1 expression and thereby might contribute to the predisposing mechanism. Additional molecular mechanisms causing ASE could be nonsense-mediated mRNA decay, due to a mutation in the target gene, which seems to be a rare event. Another possible mechanism could be the modulation of miRNA binding due to sequence variations or sequence alterations affecting the promoter activity of cancer genes. High throughput genome analysis hasAllele-Specific Expression of DAPK1 in CLLuncovered copy number variations occurring throughout genomes 1531364 of healthy individuals. These variations could result in allelic imbalances of gene expression. Similarly, differentially methylated regions other than at imprinted regions can have similar effects on gene expression. The extent to which these mechanisms participate in ASE and cancer predisposition needs to be determined in future studies. Furthermore, prospective trials are needed to confirm these findings and to extend a predisposing role of DAPK1 ASE to non-malignant CLL precursor states like monoclonal Bcell lymphocytosis.ligated PCR products of the DAPK1 cDNA. rs1056719 indicates the polymorphic site, the arrows represent the cloning primers and indicate the sequencing direction. (B) Chromatograms representing the genomic region around the polymorphic site rs1056719 in Granta-519 cells. The upper panel displays the cDNA, the lower panel displays the genomic DNA as balancing control. (C) Chromatograms representing the polymorphic site rs3818584 in EHEB cells according to figure 3B. (TIF)Figure S6 Re-balancing of DAPK1 mRNA expression inSupporting InformationFigure S1 Accurate quantification of DAPK1 ASE investigating all four common exonic SNPs. Standard curves for plasmid based standards displaying allelic ratios from 1:50 to 50:1 and correlation with idealized ratios. (A) SNP rs3118863, DAPK1 exon 26, (B) SNP rs3818584, DAPK1 exon 16, (C) SNP rs36207428, DAPK1 exon 3. (D) SNP rs1056719, DAPK1 exon 26. (TIF) Figure S2 Detection sensitivity for quantitative genotyping of rs1056719. (A) Standard curves for plasmid molecular standard (A: 30,000 template plasmid copies, B: 300 template plasmid copies) and comparison with ideal linear correlation. Standard deviations are given for 4 replicate measurements. (TIF) Figure S3 DAPK1 ASE in CD19 depleted PBMC samples from CLL patients. (A) 120 CLL cases, 11 CD19 depleted (contaminating CD19+ population less than 2 ) 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 were measured with the outlined SNuPE/MALDI-TOF-based method. (TIF) Figure S4 MEC-1 cells are fully methylated at the CpG island of the DAPK1 59 region. (A) Scheme of the DAPK1 promoter region and the associated CpG island. Grey boxes display the first 2 exons of DAPK1. Nucleotide positions are given relative to the DAPK1 transcriptional start site. Dashed lines represent positions of investigated regions/amplicons. (B) Quantitative DNA methylation analysis of the DAPK1 gene 59 region (amplicons A ) in untreated, control (PBS)-treated and 5-aza-29deoxycytidine (DAC)-treated MEC-1 cells was performed using the MassCleave method. Bars represent quantitative DNA methylation values ( ) at single CpG units. (TIF) Figure S5 Detection of DAPK1 ASE by conventionalGranta-519 cells upon inhibition of DNA methyltransferas.
