ooggeenneessii iinn ss && rrcc aa MM CC uu tt ff aa Journal ofJournal of oo gg Aya-Bonilla et al., J Carcinog Mutagen 2014, 5:6 ll ee ee aa aa nn nn nn nn ee ee rr rr ss ss uu uu ii ii ss ss oo oo DOI: 10.4172/2157-2518.1000195 JJ JJ ISSN: 2157-2518 CarCarcinogenesiscinogenesis & Mutagenesis Research Article Article OpenOpen Access Access Fine Mapping of the 15q21 Region Implicates TP53BP1 and B2M in the Lymphomagenesis of Follicular and Diffuse Large B-Cell Lymphomas Aya-Bonilla Carlos1, Camilleri Emily1,2, Benton Miles1, Haupt Larisa M1, Marlton Paula3, Lea Rod A1, Gandhi Maher K3,4 and Griffiths Lyn R1* 1Genomics Research Centre, Institute of Health and Biomedical Innovation, Queensland University of Technology, Qld Australia 2Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, USA 3Department of Haematology, Princess Alexandra Hospital, Woollongabba, Australia 4Centre for Experimental Haematology, Translational Research Institute, Woollongabba, Australia Abstract Background and aim: Recent studies focusing on the discovery of common alterations across cases suffering from follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) have reported loss of heterozygosity (LOH) and deletions events targeting the 15q21 region, indicating the relevance of this region in the lymphomagenesis of FL and DLBCL. Herein, we investigated the genetic structure of this region by studying identified LOH and copy-loss events and examined this region in the lymphomagenesis of FL and DLBCL. Methods: Fine mapping of the genomic region between the 15q15.1 and 15q21.1 loci was performed using data from copy number variation (CNV) and high resolution LOH analyses of FL (n=21) and DLBCL (n=21) cases. Validation of LOH of this region was performed using microsatellites followed by quantitative-PCR (qPCR) to measure the transcriptional abundance of TP53BP1 and B2M. Also, direct sequencing of exons 1 and 2 of B2M was performed on tumor DNA from 24 FL and 23 DLBCL samples. Results: The integration of LOH and CNV data identified copy-loss alterations at the 15q21 loci spanning a 7.5 Mb region, covering two LOH regions, termed LOH-1 and LOH-2. The LOH-1 region spans 3.4 Mb and contains 53 genes, from which TP53BP1 (tumor-protein-p53-binding-protein-1) and B2M (Beta-2-Microglobulin) were identified as the most likely target genes due to their roles in DNA double strand break (DSB) repair and immune recognition, respectively. Expression analyses revealed a significant up-regulation of TP53BP1 in NHL with LOH, but no significant changes inB2M expression were observed. Direct sequencing of exons 1 and 2 in B2M in FL and DLBCL identified two monoallelic microdeletions associated with DLBCL. Conclusion: This study identified that deletion mapping to the 15q21 locus cover two LOH regions. LOH of the TP53BP1 and B2M genes appear to be common alterations in FL and DLBCL tumorigenesis. Keywords: LOH; Copy-loss events; TP53BP1; B2M; DLBCL and FL identification and study of inactivated TSGs by copy-loss (hemizygous lymphomagenesis deletions) or copy-neutral events (chromosomal duplications, genomic or epigenetic mutations) [8,9]. The recent use of SNP array data from Introduction an unpaired cohort of tumors has enabled the inference of LOH events Follicular lymphoma (FL) and diffuse large B-cell lymphoma in tumor samples without the need for comparison to their normal (DLBCL) are responsible for over 60% of newly diagnosed cases of counterparts [8,10]. Non-Hodgkin’s lymphoma (NHL) [1,2]. FL and DLBCL, have indolent Using high resolution LOH analysis, we recently reported LOH and aggressive clinical courses, respectively; however, they share the in common regions across FLs and DLBCLs including candidate same origin (Germinal B-cell centre), but exhibit high heterogeneity tumor suppressor genes (TSGs) within these regions [11]. The precise in their morphology, clinical outcome and genetic makeup. identification of genes within these commonly affected regions Approximately 20% of FL cases transform to DLBCL, characterised is relevant for a better understanding of the common pathogenic by the acquisition of a wide array of secondary alterations, including structural genomic alterations (i.e. translocations including c-MYC, mechanisms that may play a role in the lymphomagenesis of FL and BCL6 genes) and genetic mutations (e.g. TP53), following constitutive DLBCL. One of the most common LOH regions, in FL and DLBCL BCL-2 overexpression, as a result of the translocation, t(14;18) (q32;q21) [3-6]. Nonetheless, despite differences in genetic aberrations underlying the diverse tumor biology of FL and DLBCL, the presence *Corresponding author: Prof. Lyn R. Griffiths, Institute of Health and of common genomic alterations between FL and DLBCL indicates that Biomedical Innovation, Queensland University of Technology, Musk Ave, Kelvin Grove QLD, 4059, Australia, Tel: +617 3138 6102; Fax: +617 3138 6039; lymphomagenesis of FL and DLBCL may be caused by inactivation E-mail: [email protected] or activation of shared genes and pathways, which can be crucial for Received September 25, 2014; Accepted October 27, 2014; Published October survival and development of these B-cell lymphomas [7]. 31, 2014 The identification of novel tumor suppressor genes (TSGs) and Citation: Carlos AB, Emily C, Miles B, Larisa MH, Paula M, et al. (2014) their role in oncogenesis is paramount for a better understanding Fine Mapping of the 15q21 Region Implicates TP53BP1 and B2M in the of the etiology and pathogenesis of cancer. TSGs negatively regulate Lymphomagenesis of Follicular and Diffuse Large B-Cell Lymphomas. J Carcinog Mutagen 5: 195. doi:10.4172/2157-2518.1000195 vital pathways for the proliferation and sustenance of the malignant phenotype of cancer cells; as such, they are often targets of genomic Copyright: © 2014 Carlos AB, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted and genetic alterations that disrupt their function to trigger oncogenic use, distribution, and reproduction in any medium, provided the original author and processes. Loss of heterozygosity (LOH) analysis is a useful tool for the source are credited. J Carcinog Mutagen ISSN: 2157-2518 JCM, an open access journal Volume 5 • Issue 6 • 1000195 Citation: Carlos AB, Emily C, Miles B, Larisa MH, Paula M, et al. (2014) Fine Mapping of the 15q21 Region Implicates TP53BP1 and B2M in the Lymphomagenesis of Follicular and Diffuse Large B-Cell Lymphomas. J Carcinog Mutagen 5: 195. doi:10.4172/2157-2518.1000195 Page 2 of 8 cases, spanned the chromosomal bands 15q15.1 and 15q21 [11]. of all genes located within the LOH-1 and LOH-2 regions was LOH of this genomic region has also been described in pediatric performed to investigate and overlap these LOH genes with collections glioblastomas [12]. Several alterations within and surrounding this of curated gene sets, including canonical pathways, Biocarta, KEGG, region has also been described in B-cell lymphomas. In a previous and Reactome, from the Molecular Signature Database (MsigDB, v3.1). study, integrating copy number and gene expression data analyses, we The significance level for this analytical approach was set at α<0.05 with identified that loss of 15q21 was a shared alteration between FLs and a FDR <0.05 DLBCLs [7]. Additionally, deletion of both 15q and 8p was correlated with poor prognosis in DLBCL cases, and loss of 15q23 has been TP53BP1 and B2M expression analyses considered as a secondary hit in the transformation of FL to DLBCL TP53BP1 and B2M mean fluorescence intensity values from an [3,13]. Furthermore, a previous paired high resolution LOH study Illumina Sentrix Human-6 (v2.0) array of 14 FLs and 17 DLBCLs with identified a 70 Mb region (15q13.3-15q26.3) as a target of LOH-driving available LOH data were analyzed as previously described [11]. In copy-neutral events in FL [14]. order to investigate the effect LOH on TP53BP1 and B2M expression, Hence, based on the discovery that the deletion of 15q21 and LOH TP53BP1 or B2M expression was compared between cases with RET of the region 15q15.1-15q21 are common alterations across FLs and (retention) and LOH. Cell of origin (COO) subclassification of DLBCL DLBCLs and the potential for TSGs located within this region to play a samples was based on criteria established by Shipp [22], resulting in 10 crucial role in the lymphomagenesis of these B-cell lymphomas [7,11], out 21 DLBCL cases classified as GCB-like subtype and 11 out of 21 as we undertook copy number and LOH analyses, integrating this data non-GCB-like subtype. for fine-mapping of this region. This approach was used to determine Transcript abundance of TP53BP1 and B2M was measured by overlapping regions affected by LOH and copy-loss events in these quantitative PCR (qPCR). Briefly, cDNA was reverse transcribed NHL subtypes, and to identify candidate TSGs targeted by these events. from total RNA isolated from tonsil samples of 6 healthy individuals By undertaking this integrative approach, we identified that the region and from 41 tumor tissues with high resolution LOH data as previ- targeted by copy-loss events spans 7.5 Mb and contains two specific ously described [11]. Expression levels of TP53BP1 and B2M were de- LOH regions, named as LOH-1 and LOH-2, which span a region of termined by normalization with to RPL13A using the relative quan- 3.4 Mb and 1.1 Mb, respectively. We then focused our studies on the tification (2-∆∆Ct) method. The oligonucleotide sequences used were: LOH-1 region and identified the TP53BP1 (tumor protein p53 binding TP53BP1-F (5’-TTGCCATGCCAACCAGCTCCAG-3’), TP53BP1-R protein 1) and B2M (Beta-2-Microglobulin) genes as commonly (5’-TTCACGGGGTTGCCAGTCCAG-3’), B2M-F (5’-TGCCTGCC- inactivated TSGs in FLs and DLBCLs.