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Identification and molecular characterization of recurrent genomic deletions on 7p12 in the IKZF1 gene in a large cohort of BCR-ABL1-positive acute lymphoblastic leukemia patients: on behalf of Gruppo Italiano Malattie Ematologiche dell'Adulto Acute Leukemia Working Party (GIMEMA AL WP)

The BCR-ABL1 fusion gene defines the subgroup of acute lymphoblastic leukemia (ALL) with the worst clinical prognosis. To identify oncogenic lesions that combine with BCR-ABL1 to cause ALL, we used Affymetrix Genome-Wide Human SNP arrays (250K NspI and SNP 6.0), fluorescence in situ hybridization, and genomic polymerase chain reaction to study 106 cases of adult BCR-ABL1-positive ALL. The most frequent somatic copy number alteration was a focal deletion on 7p12 of IKZF1, which encodes the transcription factor Ikaros and was identified in 80 (75%) of 106 patients. Different patterns of deletions occurred, but the most frequent were those characterized by a loss of exons 4 through 7 (Delta4-7) and by removal of exons 2 through 7 (Delta2-7). A variable number of nucleotides (patient specific) were inserted at the conjunction and maintained with fidelity at the time of relapse. The extent of the Delta4-7 deletion correlated with the expression of a dominant-negative isoform with cytoplasmic localization and oncogenic activity, whereas the Delta2-7 deletion resulted in a transcript lacking the translation start site. The IKZF1 deletion also was identified in the progression of chronic myeloid leukemia to lymphoid blast crisis (66%) but never in myeloid blast crisis or chronic-phase chronic myeloid leukemia or in patients with acute myeloid leukemia. Known DNA sequences and structural features were mapped along the breakpoint cluster regions, including heptamer recombination signal sequences recognized by RAG enzymes during V(D)J recombination, suggesting that IKZF1 deletions could arise from aberrant RAG-mediated recombination.

Gene amplification as double minutes or homogeneously staining regions in solid tumors: Origin and structure

Double minutes (dmin) and homogeneously staining regions (hsr) are the cytogenetic hallmarks of genomic amplification in cancer. Different mechanisms have been proposed to explain their genesis. Recently, our group showed that the MYC-containing dmin in leukemia cases arise by excision and amplification (episome model). In the present paper we investigated 10 cell lines from solid tumors showing MYCN amplification as dmin or hsr. Particularly revealing results were provided by the two subclones of the neuroblastoma cell line STA-NB-10, one showing dmin-only and the second hsr-only amplification. Both subclones showed a deletion, at 2p24.3, whose extension matched the amplicon extension. Additionally, the amplicon structure of the dmin and hsr forms was identical. This strongly argues that the episome model, already demonstrated in leukemias, applies to solid tumors as well, and that dmin and hsr are two faces of the same coin. The organization of the duplicated segments varied from very simple (no apparent changes from the normal sequence) to very complex. MYCN was always overexpressed (significantly overexpressed in three cases). The fusion junctions, always mediated by nonhomologous end joining, occasionally juxtaposed truncated genes in the same transcriptional orientation. Fusion transcripts involving NBAS (also known as NAG), FAM49A, BC035112 (also known as NCRNA00276), and SMC6 genes were indeed detected, although their role in the context of the tumor is not clear.

Independent centromere formation in a capricious, gene-free domain of chromosome 13q21 in Old World monkeys and pigs

BackgroundEvolutionary centromere repositioning and human analphoid neocentromeres occurring in clinical cases are, very likely, two stages of the same phenomenon whose properties still remain substantially obscure. Chromosome 13 is the chromosome with the highest number of neocentromeres. We reconstructed the mammalian evolutionary history of this chromosome and characterized two human neocentromeres at 13q21, in search of information that could improve our understanding of the relationship between evolutionarily new centromeres, inactivated centromeres, and clinical neocentromeres.ResultsChromosome 13 evolution was studied, using FISH experiments, across several diverse superordinal phylogenetic clades spanning >100 million years of evolution. The analysis revealed exceptional conservation among primates (hominoids, Old World monkeys, and New World monkeys), Carnivora (cat), Perissodactyla (horse), and Cetartiodactyla (pig). In contrast, the centromeres in both Old World monkeys and pig have apparently repositioned independently to a central location (13q21). We compared these results to the positions of two human 13q21 neocentromeres using chromatin immunoprecipitation and genomic microarrays.ConclusionWe show that a gene-desert region at 13q21 of approximately 3.9 Mb in size possesses an inherent potential to form evolutionarily new centromeres over, at least, approximately 95 million years of mammalian evolution. The striking absence of genes may represent an important property, making the region tolerant to the extensive pericentromeric reshuffling during subsequent evolution. Comparison of the pericentromeric organization of chromosome 13 in four Old World monkey species revealed many differences in sequence organization. The region contains clusters of duplicons showing peculiar features.

Gene expression profile analysis in human T lymphocytes from patients with Down Syndrome.

Down Syndrome (DS) is caused by the presence of three copies of the whole human chromosome 21 (HC21) or of a HC21 restricted region; the phenotype is likely to have originated from the altered expression of genes in the HC21. We apply the cDNA microarray method to the study of gene expression in human T lymphocytes with trisomy 21 in comparison to normal cells.

Evolutionary-new centromeres preferentially emerge within gene deserts

BackgroundEvolutionary-new centromeres (ENCs) result from the seeding of a centromere at an ectopic location along the chromosome during evolution. The novel centromere rapidly acquires the complex structure typical of eukaryote centromeres. This phenomenon has played an important role in shaping primate karyotypes. A recent study on the evolutionary-new centromere of macaque chromosome 4 (human 6) showed that the evolutionary-new centromere domain was deeply restructured, following the seeding, with respect to the corresponding human region assumed as ancestral. It was also demonstrated that the region was devoid of genes. We hypothesized that these two observations were not merely coincidental and that the absence of genes in the seeding area constituted a crucial condition for the evolutionary-new centromere fixation in the population.ResultsTo test our hypothesis, we characterized 14 evolutionary-new centromeres selected according to conservative criteria. Using different experimental approaches, we assessed the extent of genomic restructuring. We then determined the gene density in the ancestral domain where each evolutionary-new centromere was seeded.ConclusionsOur study suggests that restructuring of the seeding regions is an intrinsic property of novel evolutionary centromeres that could be regarded as potentially detrimental to the normal functioning of genes embedded in the region. The absence of genes, which was found to be of high statistical significance, appeared as a unique favorable scenario permissive of evolutionary-new centromere fixation in the population.

Evolutionary descent of a human chromosome 6 neocentromere: A jump back to 17 million years ago

Molecular cytogenetics provides a visual, pictorial record of the tree of life, and in this respect the fusion origin of human chromosome 2 is a well-known paradigmatic example. Here we report on a variant chromosome 6 in which the centromere jumped to 6p22.1. ChIP-chip experiments with antibodies against the centromeric proteins CENP-A and CENP-C exactly defined the neocentromere as lying at chr6:26,407-26,491 kb. We investigated in detail the evolutionary history of chromosome 6 in primates and found that the primate ancestor had a homologous chromosome with the same marker order, but with the centromere located at 6p22.1. Sometime between 17 and 23 million years ago (Mya), in the common ancestor of humans and apes, the centromere of chromosome 6 moved from 6p22.1 to its current location. The neocentromere we discovered, consequently, has jumped back to the ancestral position, where a latent centromere-forming potentiality persisted for at least 17 Myr. Because all living organisms form a tree of life, as first conceived by Darwin, evolutionary perspectives can provide compelling underlying explicative grounds for contemporary genomic phenomena.

Genomic organization and evolution of double minutes/homogeneously staining regions with MYC amplification in human cancer

The mechanism for generating double minutes chromosomes (dmin) and homogeneously staining regions (hsr) in cancer is still poorly understood. Through an integrated approach combining next-generation sequencing, single nucleotide polymorphism array, fluorescent in situ hybridization and polymerase chain reaction-based techniques, we inferred the fine structure of MYC-containing dmin/hsr amplicons harboring sequences from several different chromosomes in seven tumor cell lines, and characterized an unprecedented number of hsr insertion sites. Local chromosome shattering involving a single-step catastrophic event (chromothripsis) was recently proposed to explain clustered chromosomal rearrangements and genomic amplifications in cancer. Our bioinformatics analyses based on the listed criteria to define chromothripsis led us to exclude it as the driving force underlying amplicon genesis in our samples. Instead, the finding of coexisting heterogeneous amplicons, differing in their complexity and chromosome content, in cell lines derived from the same tumor indicated the occurrence of a multi-step evolutionary process in the genesis of dmin/hsr. Our integrated approach allowed us to gather a complete view of the complex chromosome rearrangements occurring within MYC amplicons, suggesting that more than one model may be invoked to explain the origin of dmin/hsr in cancer. Finally, we identified PVT1 as a target of fusion events, confirming its role as breakpoint hotspot in MYC amplification.

Genomic segmental duplications on the basis of the t(9;22) rearrangement in chronic myeloid leukemia

A crucial role of segmental duplications (SDs) of the human genome has been shown in chromosomal rearrangements associated with several genomic disorders. Limited knowledge is yet available on the molecular processes resulting in chromosomal rearrangements in tumors. The t(9;22)(q34;q11) rearrangement causing the 5′BCR/3′ABL gene formation has been detected in more than 90% of cases with chronic myeloid leukemia (CML). In 10–18% of patients with CML, genomic deletions were detected on der(9) chromosome next to translocation breakpoints. The molecular mechanism triggering the t(9;22) and deletions on der(9) is still speculative. Here we report a molecular cytogenetic analysis of a large series of patients with CML with der(9) deletions, revealing an evident breakpoint clustering in two regions located proximally to ABL and distally to BCR, containing an interchromosomal duplication block (SD_9/22). The deletions breakpoints distribution appeared to be strictly related to the distance from the SD_9/22. Moreover, bioinformatic analyses of the regions surrounding the SD_9/22 revealed a high Alu frequency and a poor gene density, reflecting genomic instability and susceptibility to rearrangements. On the basis of our results, we propose a three-step model for t(9;22) formation consisting of alignment of chromosomes 9 and 22 mediated by SD_9/22, spontaneous chromosome breakages and misjoining of DNA broken ends.

Hominoid chromosomal rearrangements on 17q map to complex regions of segmental duplication

BackgroundChromosomal rearrangements, such as translocations and inversions, are recurrent phenomena during evolution, and both of them are involved in reproductive isolation and speciation. To better understand the molecular basis of chromosome rearrangements and their part in karyotype evolution, we have investigated the history of human chromosome 17 by comparative fluorescence in situ hybridization (FISH) and sequence analysis.ResultsHuman bacterial artificial chromosome/p1 artificial chromosome probes spanning the length of chromosome 17 were used in FISH experiments on great apes, Old World monkeys and New World monkeys to study the evolutionary history of this chromosome. We observed that the macaque marker order represents the ancestral organization. Human, chimpanzee and gorilla homologous chromosomes differ by a paracentric inversion that occurred specifically in the Homo sapiens/Pan troglodytes/Gorilla gorilla ancestor. Detailed analyses of the paracentric inversion revealed that the breakpoints mapped to two regions syntenic to human 17q12/21 and 17q23, both rich in segmental duplications.ConclusionSequence analyses of the human and macaque organization suggest that the duplication events occurred in the catarrhine ancestor with the duplication blocks continuing to duplicate or undergo gene conversion during evolution of the hominoid lineage. We propose that the presence of these duplicons has mediated the inversion in the H. sapiens/P. troglodytes/G. gorilla ancestor. Recently, the same duplication blocks have been shown to be polymorphic in the human population and to be involved in triggering microdeletion and duplication in human. These results further support a model where genomic architecture has a direct role in both rearrangement involved in karyotype evolution and genomic instability in human.

t(3;12)(q26;q14) in polycythemia vera is associated with upregulation of the HMGA2 gene

Polycythemia vera (PV) is a chronic myeloproliferative disorder (MPD) characterized by an excess production of apparently normal erythrocytes and a variable overproduction of leukocytes and platelets in the absence of a defined cause. Recent PV studies have identified in several patients an acquired valine-tophenylalanine mutation at amino acid 617 (V617F) in the Janus kinase 2 (JAK2) tyrosine kinase gene. This mutation, however, is not present in all PV patients. Other studies have reported a number of gene fusions resulting from chromosomal aberrations, but no recurrent chromosomal abnormality has been identified so far. We report here a PV case showing a novel, balanced t(3;12)(q26;q14) translocation, involving the 30 UTR of the gene HMGA2 (high mobility group AT-hook 2 isoform a) and the coding region of the TNIK (Traf2and Nck-interacting kinase). Molecular analyses demonstrated an overexpression of HMGA2, which, however, was not due to the formation of a fusion transcript with TNIK, but, very likely, to a position effect. TNIK expression pattern was not affected by the rearrangement. A 68-year-old man presented in February 2002 with polycythemia, and a 60% hematocrit (Hct) level. Bone marrow trephine biopsy showed a hypercellular marrow with an increased erythroid series. Molecular analysis revealed homozygosity for JAK2. In accordance with polycythemia vera study group (PVSG) criteria, a diagnosis of PV was made. The patient was treated with phlebotomy, performed every 2–3 months, and hydroxyurea. To date, he is alive and periodically undergoes phlebotomy. Bone marrow (BM) karyotype revealed a translocation 46,XY,t(3;12)(q26;q14)[11] (Figure 1a). Multicolor-fluorescent in situ hybridization (FISH), performed using the commercially available 24-colour SpectraVysion probe (Abbott, Abbott Park IL, USA), confirmed the t(3;12) rearrangement (Figure 1a). FISH experiments using appropriate BAC clones, identified in the UCSC database (www.genome.ucsc.edu/March 2006 release), revealed that the breakpoints were located within BAC clones RP11–466C5 (chr3:172 251 934–172 458 321, at 3q26.2; Figure 1a and b), and RP11–366L20 (chr12:64 532 786–64 711 531, at 12q14.3; Figure 1a and b). The sequence encompassed by the latter BAC was found to contain only the gene HMGA2. The breakpoint region on chromosome 3 was also found to contain a single gene, TNIK, encoding a stress-activated serine/threonine kinase. To further narrow the breakpoint within this gene, we generated a pool of long-polymerase chain reaction (PCR) products (TN2–TN7) spanning the genomic region from intron 3 to intron 12 (chr3: 172 344 335–172 417 421; Figure 1a and b; the pairs of primers used to obtain the LONG-PCR fragments are listed in Table 1). The probe pool detected, in addition to a signal on the normal chromosome 3, two splitting signals on der(3) and der(12), thus indicating that the breakpoint was located inside TNIK (Figure 1a and b). A LONG-PCR experiment using a forward primer specific for exon 5 of the HMGA2 gene (Ex5aF, Table 1) and the reverse primer TNIK4R from the TNIK gene (Table 1), designed to amplify the junction region on der(12), generated a fragment of B8.5 kb (data not shown). Nested PCR experiments with the primer combination Ex5aF and TNIK4.1R (6757 bp centromeric to TNIK4R, Table 1) yielded a PCR product of 1780 bp. Both PCR experiments failed to produce any amplification fragment in control experiments using the DNA from a healthy individual (Figure 1c). The 1780 bp fragment was entirely sequenced. Sequence analysis showed that chromosome 12, at nucleotide (nt) 64 644 064 (exon 5 of HMGA2), was fused with chromosome 3 at nt 172 374 322 (intron 9 of TNIK) (Figure 1d). The reciprocal genomic junction TNIK/HMGA2 was also PCR amplified using the primer set TNIK2BR.F/EX5aR. The resulting 1130 bp fragment (Figure 1c) was sequenced. It revealed that chromosome 3, at nt 172 374 440 (intron 9 of TNIK), was fused with chromosome 12 at nt 64 643 497 (exon 5 of HMGA2) (Figure 1e). Sequence comparison of the two breakpoint regions, using the BLAT tool of the UCSC Genome Browser, revealed the occurrence of 118 and 567 bp nucleotide overlaps, respectively, within TNIK intron 9 and HMGA2 exon 5 (data not shown). Moreover, nucleotide microhomologies of two nucleotides (AG) and six nucleotides (ATCACAT) were detected at the translocation junctions, respectively, on chromosomes der(12) and der(3) (Figure 1d and e). Although the two genes appeared fused at the genomic level, 30 rapid amplification of cloned ends (RACE)PCR (Clontech/BD Biosciences, Mountain View, CA, USA) experiments excluded the presence of a 50HMGA2/30TNIK chimeric transcript (data not shown). The HMGA2 and TNIK gene expression level was determined by real-time PCR using 1 PlatinumSYBR Green qPCR SuperMix-UDG (Invitrogen, Carlsbad, CA, USA). The gene expression level variation was estimated by comparing the values of 2 DDCt (relative amount of cDNA) for both the HMGA2 and TNIK genes’ Ct values in the patient with t(3;12) with the relative value of a control, obtained considering a Ct mean value. The significance was estimated by comparing respective ranges ð2 DDCt s:d:Þ of these values (Figure 1f and g). 28S rRNA was used as reference gene and one (No. 6, Figure 1f and g) of the six PV cases with a normal karyotype (controls), included in the study, was utilized as calibrator. Real-time PCR analyses of the HMGA2 and TNIK expression levels revealed upregulation of both HMGA2 exon 3 and exon 5 (Figure 1f), but no noteworthy changes in the expression level of TNIK (Figure 1g) were detected. For the latter gene, we used primers specific for regions both upstream (exons 2–3, 8–9) and downstream (exons 10, 11–12, 17–18) of the breakpoint (primer sequences are available on request). In summary, our study have indicated that the HMGA2 full transcript (exons 1–5) level was higher in the patient bearing the t(3;12) translocation than in other PV cases with a normal karyotype (Figure 1f). The upregulation of HMGA2 was likely due to a position effect. This phenomenon has already been documented, by our and other groups, in hematological disorders involving the EVI1 gene. In fact, the breakpoint, at the genomic level, was mapped within the 30UTR of the gene, a region containing regulatory elements that exert posttranscriptional expression control. It can be, therefore, hypothesized that the removal of this gene segment alters the expression pattern of HMGA2 by upregulation of the ‘wild-type’ Letters to the Editor