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Blood, Vol. 113, Issue 20, 4918-4921, May 14, 2009
The NF- B negative regulator TNFAIP3 (A20) is inactivated by somatic mutations and genomic deletions in marginal zone lymphomas
Blood Novak et al.
113: 4918
Supplemental materials for: Novak et al
Phenotypic characterization
Immunohistochemical (IHC) staining was performed with an automated staining machine (Universal staining system, DAKO) after heat induced antigen retrieval, as applicable. Primary antibodies included CD21, CD23, CD20, CD79a, CD3, CD5, BCL2, BCL6, CD10, cyclin D1, MUM1, and MIB-1 (DAKO, Carpinteria, CA, USA). The Envision Plus kit (DAKO) and chromogen 3,3′-diaminobenzidine were used for detection. Three or four-color flow cytometric analysis was performed using FACScan (Becton Dickinson, San Diego, CA, USA) according to standard methods and data was analyzed with the Cell Quest software (Becton Dickinson). The antibodies used included: CD45, CD43, CD19, CD20, CD79a, CD10, CD23, FMC7, CD11c, kappa, lambda, CD2, CD3, CD4, CD5, CD7, CD8, and CD16/56 (Becton Dickinson, San Diego, CA, USA). Genome-wide DNA-profiling (SNP-array analysis)
Genomic DNA integrity was verified by agarose-gel electrophoresis.1 Genome-wide DNA profiles were obtained using the GeneChip Human Mapping 250K NspI and StyI Arrays (Affymetrix, Santa Clara, CA, USA), following the manufacturer’s protocol and analyzed as previously described.1 Briefly, data acquisition was performed using the Affymetrix GCOS 1.4 and GTYPE 4.1. Genotype calls were calculated using the BRLMM algorithm within a data set of over 100 B-cell tumors analyzed in house to improve the genotype call. The Affymetrix CNAT 4.01 was used to calculate copy-numbers (CN) starting from the BRLMM-CHP files. Analyses were performed using CN Gaussian bandwidth of 100 Kb and LOH transition decay of 10Mb. The CNAT hidden Markov model (HMM) was used to estimate CN. Samples were normalized using 46 Caucasian normal female samples of the HapMap Project. CN gains and losses were defined in the presence of CN above 2.15 and CN below 1.85, respectively, corresponding to a p-value of < 0.001 after Bonferroni multiple test correction. LOH profiles were obtained applying the method with haplotype correction for tumor-only LOH inference available in the dChip software, using as reference the 60 CEPH parents of the HapMap Project and computing the allele A frequency from the data. Heat-maps were built by plotting the median Log2 ratio values of CNAT HMM CN estimate with the 100 Kb Gaussian bandwidth, and the loss versus retention LOH call from dCHIP. Methylation of the TNFAIP3 (A20) promoter (Data not shown)
Methylation of the TNFAIP3 promoter was analyzed using bisulfite sequencing PCR. The 1000 bp DNA sequence upstream from the start of TNFAIP3 (NM_006290) exon 1 as obtained from the UCSC Genome Browser.2 Bisulfite-treatment of 600 ng of genomic DNA was done using the EpiTect Bisulfite Kit (Qiagen, Hombrechtikon, Switzerland) according to the manufacturer’s protocol. Forward (5′-AGATGGGGATTAAAGGTTGTTAT-3′) and reverse (5′-CAAAAAATCATCAACTATAAAAAAAA-3′) primers were selected using the MethPrimer software.3 Cycling parameters were: 95°C for 3′ min, followed by 40 cycles at 95°C for 30”, 52.5°C for 30”, 72°C for 40”, and final extension at 72°C for 7′. PCR products were purified using the QiAquick PCR Purification Kit (Qiagen, Hombrechtikon, Switzerland). Direct DNA sequencing was performed using an external service (Microsynth AG, Balgach, Switzerland). REFERENCES 1. Forconi F, Poretti G, Kwee I, et al. High density genome-wide DNA profiling reveals a remarkably stable profile in hairy cell leukaemia. Br J Haematol. 2008;141:622-630.
2. Karolchik D, Hinrichs AS, Furey TS, et al. The UCSC Table Browser data retrieval tool. Nucleic Acids Res. 2004;32:D493-496.
3. Li LC, Dahiya R. MethPrimer: designing primers for methylation PCRs. Bioinformatics. 2002;18:1427-1431.
Files in this Data Supplement:
- Table S1. Clinicopathologic characterization of marginal zone B-cell lymphomas (XLS, 60 KB)
- Table S2. Detailed A20 sequencing results (XLS, 62.5 KB)
- Table S3. Genetic loss of the A20 gene locus by FISH and SNP-array analyses (XLS, 51 KB)
- Table S4. PCR primers and protocols (XLS, 50.5 KB)
- Figure S1. SNP-array analysis of 29 MZLs (10 ENMZL: 4 parotid, 3 lung, 1 skin, 1 jejunum, 1 orbital, 9 NMZL, 10 SMZL) (JPG, 210 KB)
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(A) Frequency of chromosomal gains and losses. Upper panel: frequency of DNA gains (up/red) and losses (down/blue). Lower panel: frequency of LOH (black). X-axis, chromosome localization and physical mapping; Y-axis, percentage of cases showing genomic aberrations. (B) Heat plots summarizing gains and losses of chromosome regions per case as indicated. Upper panel: heat plot of DNA gains (red) and losses (blue). Lower panel: heat plot of LOH (black). X-axis, chromosome localization and ordered mapping; Y-axis, cases. Only lesions larger than 1 Mb are shown. Commonly gained chromosomes or chromosomal regions included chromosome 3 (5 cases), 1q21.2-q32.2 and 14q32.33 (3 cases each), chromosome 18 (2 cases, plus one with 18q only), and 11p15.5 (2 cases), while losses most frequently targeted 14q32.33 (6 cases), 15q24.1 (5 cases), 1q21.2 (4 cases), and 9p13.2 (3 cases). For 6q23 deletions, see Table S3. The complete SNP-array data have been deposited (GSE #13989, http://www.ncbi.nlm.nih.gov/geo).
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