ABSTRACT: This SuperSeries is composed of the following subset Series: GSE35424: methylation analysis was carried out on 12 SMZL, 6 MCL and 6 FL cases by the Cambridge group using Illumina Infinium Human Methylation 27 Array GSE35425: Oligo aCGH on 21 SMZL cases were carried out by the Cambridge group using the Agilent Custom 2Kb resolution Oligo aCGH GSE35426: Gene expression profiling was carried out on 14 SMZL, 5 FL and 5 MCL cases using the Affymetrix HG-U133plus2 gene expression microarrays by the Cambridge group Splenic marginal zone lymphoma (SMZL) is an indolent B-cell lymphoproliferative disorder characterised by 7q32 deletion, but the target genes of this deletion remain unknown. In order to elucidate the genetic target of this deletion, we performed an integrative analysis of the genetic, epigenetic, transcriptomic and miRNomic data. High resolution array comparative genomic hybridization of 56 cases of SMZL delineated a minimally deleted region (2.8Mb) at 7q32, but showed no evidence of any cryptic homozygous deletion or recurrent breakpoint in this region. Integrative transcriptomic analysis confirmed significant under-expression of a number of genes in this region in cases of SMZL with deletion, several of which showed hypermethylation. In addition, a cluster of 8 miRNA in this region showed under-expression in cases with the deletion, and three (miR-182/96/183) were also significantly under-expressed (P <0.05) in SMZL relative to other lymphomas. Genomic sequencing of these miRNA and IRF5, a strong candidate gene, did not show any evidence of somatic mutation in SMZL. For aCGH, 21 cases were done under accession number GSE35425. 21 fresh frozen splenic samples from patients with SMZL were hybridized against a reference control of pooled PBMC genomic DNA from 10 normal subjects. High-resolution oligonucleotide analysis of copy number variations in splenic marginal zone lymphoma (SMZL) in regions of recurrent alteration. Regions included on the array: 7q (2k resolution) and regions of interest on chromosomes 3q, 6q and 9q at 12k resolution. For gene expression microarrays, 24 cases were done under accession number GSE35426. A total of 48 cases of SMZL (including 15 with 7q deletion) were analysed using the Affymetrix HG-U133 Plus 2.0 platform (Affymetrix, Santa Clara, California, USA). Arrays were performed according to the manufacturer’s instructions. Briefly, RNA was extracted from snap frozen tissues with >70% tumour cells using the RNEasy extraction kit (Qiagen) and subjected to DNAse treatment (Turbo DNAse kit, Ambion). RNA integrity was assessed using an Agilent 2100 Bioanalyzer. cDNA synthesis was carried out with 2ug RNA using the GeneChip® One-Cycle cDNA Synthesis Kit (Affymetrix), followed by in vitro transcription with biotin-labelled nucleotides using GeneChip® IVT Labeling Kit. Biotinylated cRNA was purified and hybridized to the Affymetrix HG-U133 Plus 2.0 chips in a GeneChip® Hybridisation Oven 640 at 45oC for 14 hours. The arrays were then washed and stained using the Fluidics station 450 system (Affymetrix). The arrays were scanned using the Affymetrix GeneArray® Scanner 3000. Hybridisation and labelling controls were included according to the manufacturer’s instructions, and quality control analysis of microarrays was performed to published standards. For methylation arrays, 24 cases were done under accession number GSE35424. Epigenetic methylation analysis was performed using the Infinium Human Methylation 27 array (Illumina, San Diego, California, USA). The array contained 27,568 CpG islands within the proximal promoter regions of transcription start sites of 14,475 RefSeq genes, including 12,883 well annotated genes (NCBI CCDS database: Build 36). The methylation array was carried out in 12 SMZL (6 cases with 7q deletion), 6 follicular lymphomas (FL) and 6 mantle cell lymphomas (MCL) as per the manufacturer’s instructions. Briefly, 2μg genomic DNA extracted from frozen tissues with >70% tumour cells was bisulphite modified using the EZ DNA Methylation Kit (Zymo Research Corporation). Bisulphite modified DNA was then amplified using the MSM master mix (Illumina) and incubated at 37oC for 22 hours. Amplified DNA was then fragmented and hybridised to BeadChips in an Illumina Hybridisation Oven at 48oC for 18 hours. Following hybridisation, single base extension of hybridised DNA using hapten labelled bases was performed. Staining was then developed using immunochemical stains catalysed by the haptens, and the arrays washed. The chips were scanned using the BeadArray™ Reader (Illumina) and the BeadScan™ software (Illumina) using the Infinium Methylation Scan setting. The scanned data was then analysed in GenomeStudio™ (Illumina) using the Methylation analysis module. **Samples from Series GSE35278 and GSE35348 was also used in this study: GSE35278: Oligo aCGH on 35 SMZL cases were carried out by the Mayo clinic group using the Agilent Oligo aCGH 244A platform (6.4Kb resolution) GSE35348: Gene expression profiling was carried out by the Mayo clinic group on 24 SMZL cases using the Affymetrix HG-U133 plus2 gene expression microarrays
