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Te the DNA methylation patterns with gene expression profile and to assess the prognostic implications of such correlations on clinical outcome in 93 early stage CLL patients. MethodsPatient selectionoutlined by Rai et al. [21], 24 patients were in stage 0, 33 were in stage I and 43 were in stage II. Fourteen randomly selected CLL samples and pooled CD19+ B-cells from 10 healthy individuals were profiled for methylation. Gene expression profiling was carried out in 21 CLL samples and pooled CD19+ Bcells from 10 healthy individuals. All the CLL samples had at least 65 CLL phenotype cells. The clinical and laboratory characteristics of the CLL patients analysed using methylation and gene expression arrays are provided in Table 1. The mRNA expression of 17 of the genes identified to be differentially methylated and /or differentially expressed was validated using SYBR-green based RQ-PCR in 93 (Unmutated = 39, Mutated = 54) PRIMA-1 CLL patients. The median age of the CLL patients was 60 years (range 35?0 years). With a median follow-up time of 22 months (range 1-124 months), 46 patients required treatment [median time to treatment: 14 months (range 0?2 months)] and 18 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/22993420 patients died. On the basis of international prognostic index (IPI) score [22], 11/93 patients were assigned as low risk, 34/93 as intermediate risk, 43/93 as high risk, and 5/93 as very high risk patients.IGHV mutation statusIGHV gene family usage was evaluated as per BIOMED2 protocol [23] and the patients were assigned to IGHV mutated or unmutated subgroups based on the IGHV sequence homology (cut-off = 98 ) as determined by the international ImmunoGeneTics database (IMGT; http:// imgt.cines.fr, Montpellier, France).Methylated CpG island microarraysTreatment naive early stage (Rai 0-II) CLL patients (n = 100) were enrolled in the study after obtaining informed consent as per the guidelines of the institute ethics committee. According to the staging criteriaGenomic DNA was extracted from the peripheral blood mononuclear cells (PBMC) of CLL patients (n = 14) and CD19+ sorted cells pooled from 10 healthy individuals. To isolate the CD19+ cells, mononuclear cells isolated from peripheral blood of healthy individuals were incubated with CD19 + magnetic microbeads and processed according to the manufacturer's protocol (Milteneyi Biotech, Gladbach, Germany). In healthy individuals, CD19+ cells constitute 2-3 of the leukocyte fraction and therefore, sorted CD19+ B-cells from healthy individuals were used. In the CLL samples evaluated for microarrays, CD19+ cells constituted at least 65 of the leukocytes and the PBMC fraction from CLL patients was used for the study. For methylated CpG island microarrays, 6 g of genomic DNA was digested with Mse I restriction enzyme (New England Biolabs Inc., Ipswich, MA, USA) and labelled with anti-5 methyl cytidine antibody (Abcam, Cambridge, UK). One fraction of the labelled DNA was immunoprecipitated while the other was used as input DNA. Both the input and immunoprecipitated fractions were purified followed by whole genome amplification (WGA, Sigma Aldrich, St.Rani et al. Clinical Epigenetics (2017) 9:Page 3 ofTable 1 Clinical and laboratory characteristics of the CLL patients evaluated using methylation and gene expression arraysCharacteristics of the patients Sr. No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/9547713 20 21 22 23 24 25 26 Sample ID S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 Methylation.