Chromosomal translocations are recognized as one of the chief cause of tumour progression at molecular level that consequently develops gene fusions (Fig. 1) [1Mitelman F, Johansson B, Mertens F. The impact of translocations and gene fusions on cancer causation Nat Rev Cancer 2007; 7: 233-45.-3Gasparini P, Sozzi G, Pierotti MA. The role of chromosomal alterations in human cancer development J Cell Biochem 2007; 102: 320-1.]. These fusion genes are ideal prognostic, diagnostic markers and therapeutic targets as they attribute distinct features to specific cancer subtypes. According to earlier presumptions translocations were thought curbed primarily to hematological tumours but recent findings proposes their widespread and rising number characterizing the subset of frequent & rare solid cancers like lung, prostate, kidney, papillary thyroid carcinoma, salivary gland tumours [4Chinnaiyan AM, Palanisamy N. Chromosomal aberrations in solid tumors Prog Mol Biol Transl Sci 2010; 95: 55-94.-6Aman P. Fusion genes in solid tumors Semin Cancer Biol 1999; 9: 303-18.].

Fig. (1) Schematic representation showing mechanism of aberrant chromosomal translocations.

Fig. (2) Flow chart for retrieval of 1000 Base pair nucleotide sequences of fusion partners of major translocations from TICdb and UCSC tool.

Fig. (3) Flowchart showing multiparametric computational analysis for known breakpoint region.

Fig. (4) Identification of nearest Segmental Duplicons (SDs) by UCSC genome browser for t(21:22). This figure depicts the occurrence of SD at the genomic location (48100446-48117693) of chr 21(A) which matches with genomic segment (51226859-51244566) of chr 22 (B).

Fig. (5) Analysis of sequence motifs, destabilization sites and physico-chemical parametersin BpI region of t(21;22). (A1 & 2) It depicts occurrence of 12 & 23 bps cryptic RSSs and repetitive elements found in the BpI region comprising of the non-coding portion of the respective genes. (B1 & 2) Graphical representation of SIDD destabilization profile plotted for G(x), the incremental free energy against queried sequence. The bold arrow portrays the presence of destabilization site that is in the closest proximity to the breakpoint while the dotted arrow represents SIDD region with the highest destabilization which requires minimum energy for duplex separation. (C1 &2) Flexibility of query sequence was graphically plotted in which the line defines the high helical flexibility at the breakpoint junction (D1 &2) Graphical representation of melting temperature of query sequence was designed. The line demarcates the decreasing trend of Tm at the breakpoint junction. (E1 & 2) Higher energy levels of base stacking was found at the breakpoint junction that is denoted by a line in the pictorial representation (F1 & 2): DNA base composition analysis for SIDD region showed highest AT content at the breakpoint junction, shown by dotted line.

Like in leukemia, oncogenic fusions in epithelial cancers can be categorized into two broad group (a) tyrosine kinase e.g. papillary thyroid cancers have been characterize by RET fusion. ALK & ROS1 fusions are frequent in NSCLCs and (b) transcription factor e.g. ETV6/NTRK3 fusions are expressed mainly in secretory breast cancer While papillary thyroid carcinomas are distinguishably associated with RET and NTRK1 rearrangements. While in addition, fusions related to TMPSSR2, TFE3, PLAG1, HMGA2 are manifested with occurrence of prostate, renal, salivary gland pleiomorphic adenoma respectively [7Kaye FJ. Mutation-associated fusion cancer genes in solid tumors Mol Cancer Ther 2009; 8: 1399-408.-10Prensner Jr, Chinnaiyan AM. Oncogenic gene fusions in epithelial carcinomas Curr Opin Genet Dev 2009; 19: 82-91.] (Fig. F1). Assessment of these rearrangements which directs the formation of breakpoints has divulged numerous recurring thoughts providing crucial depth into mechanism of carcinogenesis.

The issue of prime focus is the precision with which the chromosomes breaks & reunites within genome and in order to achieve this, one key feature of particular importance is to study the genomic sequence lying in the vicinity of breakpoints. This will explicate probable role of the genomic architecture and will also define what those potential feature may be. For this, functional annotation are required to be accompanied with physical information to understand the structure, dynamics and the common functionality of genomic DNA, due to which prevalence of breakpoints may be associated with several genomic features.

From previous records it has been emphasized that chromatin structural elements are associated with and responsible for double strand breaks within genome which are incorrectly ligated resulting into recurring chromosomal translocation [11Albano F, Anelli L, Zagaria A. Non-random distribution of genomic features in breakpoint regions involved in chronic myeloid leukemia cases with variant t(9,22):or additional chromosomal rearrangements Mol Cancer 2010; 9: 120.-13Novo FJ, Vizmanos JL. Chromosome translocations in cancer computational evidence for the random generation of double-strand breaks Trends Genet 2006; 22: 193-6.]. Therefore, in order to attain profound knowledge of the molecular mechanism of carcinogenesis and a holistic idea concerning the behavioral patterns of these breakpoints, we contemplated to scrutinize a variety of factors including segmental duplicons (SDs), destabilization profiles; Recombination signal sequences (RSS), repeats, physico-chemical characteristics of nucleic acid and many more.

Development of Innovative and sophisticated technologies for genome sequencing ended up in identifying Gene fusions as molecular signature in broad range of solid tumours, which were initially considered only to be associated with hematological tumours [20Nambiar M, Kari V, Raghavan SC. Chromosomal translocations in cancer Biochim Biophys Acta 2008; 1786: 139-52.-25Qi M, Li Y, Liu J, Yang X, Wang L, Zhou Z , et al. Morphologic features of carcinomas with recurrent gene fusions Adv Anat Pathol 2012; 19: 417-24.]. In order to explicate the cause of these chromosomal breaks, numerous potential biological mechanisms such as DNA repair by non-homologous end joining (NHEJ), Alu arbitrated homologous recombination, illegitimate V(D)J recombination and various others have been suggested [26Mridula Nambiar, Sathees C Raghavan. How does DNA break during chromosomal translocationsκ Nucleic Acids Res 2011; 39: 5813-25., 27Peter D. Aplan.Causes of oncogenic chromosomal translocation Trends Genet 2006; 22: 46-55.]. For acquisition of in- depth knowledge pertaining to these molecular mechanisms, there is requisite to punctuate that whether there is direct link between the particular pattern of local genomic sequence and breakpoint regions which may provide us with the clue for the cellular processes that promote chromosome rearrangements [12Albano F, Anelli L, Zagaria A. Genomic segmental duplications on the basis of the t(9,22):rearrangement in chronic myeloid leukemia Oncogene 2010; 29: 2509-16., 28Bacolla A, Wojciechowska M, Kosmider B, Larson JE, Wells RD. The involvement of non-BDNA structures in gross chromosomal rearrangements DNA Repair (Amst) 2006; 5: 1161-70., 29Raghavan SC, Lieber MR. DNA structures at chromosomal translocation sites Bioessays 2006; 28: 480-94.].

Substantial attention regarding the importance of Segmental Duplications in genetic dis-orders has been revealed due to significant advances in molecular cytogenetics field [30Ji Y, Eichler EE, Schwartz S, Nicholls RD. Structure of chromosomal duplicons and their role in mediating human genomic disorders Genome Res 2000; 10: 597-610., 31Emanuel BS, Shaikh TH. Segmental duplications an 'expanding' role in genomic instability and disease Nat Rev Genet 2001; 2: 791-800.]. Earlier studies describes the possible mechanisms of direct involvement of low copy repeats (LCRs) or SDs in the occurrence of t(11;22) [32Shaikh TH, Kurahashi H, Emanuel BS. Evolutionarily conserved low copy repeats (LCRs) in 22q11 mediate deletions, duplications, translocations, and genomic instability an update and literature review Genet Med 2001; 3: 6-13., 33Kolomietz E, Meyn MS, Pandita A. The role of Alu repeat clusters as mediators of recurrent chromosomal aberrations in tumors Genes Chromosomes Cancer 2002; 35: 97-112.]. Interestingly, our bio-informatic analysis, for first time demonstrates the existence of SDs flanking the breakpoint regions of solid tumor translocations and their potential role in rearrangements of DNA segments. These duplicons are found to be placed across several mega-bases, either present at common breakpoint regions or elsewhere in same sub chromosomal region. These closest duplicons are homologous sequences that search for and anchor with each other, thereby making the recombination a feasible event showing their ability to serve as substrate for aberrant genomic rearrangement.

Though, the enormous role of particular genomic architectures has been already established as casual mechanism of recurrent rearrangements but till date, this information is only confined to individual translocation cases. Hence, to the best of our knowledge, this study is a novel approach in terms of analyzing all the parameters (that may play crucial role in chromosome break) collectively for many translocations of epithelial tumour.

Enhanced evidences for the incidence of high densities of repetitive DNA sequences like Alu repeats, at translocation breakpoint regions has proposed that these sequences act as hot spots for events of recombination and thus facilitates translocation process [33Kolomietz E, Meyn MS, Pandita A. The role of Alu repeat clusters as mediators of recurrent chromosomal aberrations in tumors Genes Chromosomes Cancer 2002; 35: 97-112.-35Shaikh TH, O'Connor RJ, Pierpont ME , et al. Low copy repeats mediate distal chromosome 22q112 deletions sequence analysis predicts breakpoint mechanisms Genome Res 2007; 17: 482-91.]. Our study also revealed presence of Alu repeats in the BpIs of all translocations amongst which chromosome 22 demonstrates the highest density (82.7%) whereas control gene under investigation exhibited complete absence of such repeats favoring the verity that Alu core sequence are of prime importance in promoting DNA strand exchange and genomic rearrangement.

It is distinctly apparent from this in silico analysis that, the breakpoint junctions are situated within the intronic portion of genomic sequences. This is indicative of the fact that the presence of breakpoints in the non-coding regions will not influence the functionality of the fusion gene so produced, highlighting the point that these intronic regions so present within the genome are purposeful. Our scrutiny affirmed the preponderance of SIDD sites in BpIs or at breakpoint junction but not in control genomic sequences. This articulates that regulatory genomic sites and recombination hot spots are more prone to stress driven strand separation which has a variety of inference in replication mechanism or transcriptional regulation [36Wang H, Noordewier M, Benham CJ. Stress-induced DNA duplex destabilization (SIDD) in the E coli genome SIDD sites are closely associated with promoters Genome Res 2004; 14: 1575-84., 37Winkelmann S, Klar M, Benham C. The positive aspects of stress strain initiates domain decondensation (SIDD) Brief Funct Genomic Proteomic 2006; 5: 24-31.]. In addition, our result for all the translocation represents the existence of discrete characteristic - RSS, at or near the breakpoint region at least in either of the partner. Immense similarity is observed amongst the sequences of genes involved in solid tumour translocations and an authentic RSS sequences that normally comprises of at least a CAC, which is indispensable for RAG cleavage by V(D)J recombination thus making a double-strand break [38Zhang M, Swanson PC. V(D)J recombinase binding and cleavage of cryptic recombination signal sequences identified from lymphoid malignancies J BiolChem 2008; 283: 6717-27., 39Raghavan SC, Swanson PC, Ma Y, Lieber MR. Double-strand break formation by the RAG complex at the bcl-2 major breakpoint region and at other non-B DNA structures in vitro Mol Cell Biol 2005; 25: 5904-19.]. On the other hand, control sequences including core exonic region and intron/exon boundary showed the nonappearance of RSS, SIDD which is again in support of actuality that there is a need of exceptional phenomena for chromosomal break leading to translocation other than mechanism of splicing.

Evaluation of comprehensive data concerned to physico-chemical features depicts inverse proportionality of stability with that of flexibility and stacking energy. This noticeably imply that base stacking and helical flexibility influences protein-DNA interactions to greater extent which therefore impact upon chromatin structure and generates genomic instability at the breakpoint region responsible for chromosome breakage [40Yakovchuk P, Protozanova E, Frank-Kamenetskii MD. Base-stacking and base-pairing contributions into thermal stability of the DNA double helix Nucleic Acids Res 2006; 34: 564-74.-44Van Gent DC, Hoeijmakers JH, Kanaar R. Chromosomal stability and the DNA double-stranded break connection Nat Rev Genet 2001; 2: 196-206.]. Furthermore, comparatively high AT content is witnessed in the SIDD region as compared to whole BpIs which authenticate that AT Island are thermodynamically destabilized, exclusively flexible and remarkably prone to super helical stress thus enhancing the vulnerability of genomic breakage [45Kato T, Kurahashi H, Emanuel BS. Chromosomal translocations and palindromic AT-rich repeats Curr Opin Genet Dev 2012; 22: 221-8.-47Hiroshi Kogo, Hidehito Inagaki, Tamae Ohye. Cruciform extrusion propensity of human translocation-mediating palindromic AT-rich repeats Nucleic Acids Res 2007; 35: 1198-208.]. More to the point, perceptible discrimination amidst the breakpoint and non-breakpoint island with noteworthy specificity and sensitivity is observed, as per the cutoff values derived by ROC curve for Tm, Flexibility, AT content and stacking energy.

The in-depth knowledge derived from our computational assessment construed a straight correlation between high values stacking energy, flexibility, AT content with the presence of SIDD sites. Rest of the dynamic features though indispensable separately but are signifying discrepancy when compared with each other. Thus, these imputes are unswerving with the belief that presence of peculiar genomic patterns at or nearby breakpoints may act as driving force for DSBs causing translocations which has been illustrated in the CIRCOS plot (Fig. S2).

In summary, this multi-parametric bio-informatics methodology employed for analysis of genomic sequences of breakpoint coordinates furnishes us with superior perceptive of molecular mechanisms of DNA strand break during translocation leading to epithelial carcinogenesis. First and foremost, SDs in direct orientations are typically found close to breakpoints which might be obligatory for occurrence of efficacious recombination. Second, SIDD can be considered as a “Driver” mechanism that will help to distinguish a breakpoint region in any unidentified sequence owing to its direct connection with the physico chemical parameters as manifested from our research. Moreover, not any of the precise chromatin organization and DNA architectural motif revealed common signatures, though they are discretely imperative in increasing propensity for repeated cross over events. As our in silico investigations goes in analogue with the earlier reported in-vitro studies which eventually lead to authentication of the computational protocol followed here, reflecting the fact that chromosomal rearrangements are non-random actions which make DNA susceptible to DSBs in a precise and definite pattern. Inclusion of intron/exon flanking genomic sequences in control gene analysis strengthen the postulation that break involving translocation is inimitable by nature which is governed by manifold parameters like SIDD region, repeat, flexibility/stability, RSS, stacking energy and AT content. Furthermore this multi-parametric study can lead to the conceptualization of an algorithmic program which may predict possible breakpoint in any given sequence of human genome.