Angelo Lonoce

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.

Comparative mapping of human alphoid sequences in great apes using fluorescence in situ hybridization

Twenty-seven human alphoid DNA probes have been hybridized in situ to metaphase spreads of the common chimpanzee (PTR), the pigmy chimpanzee (PPA), and the gorilla (GGO) to investigate the evolutionary relationship between the centromeric regions of the great ape chromosomes. The surprising results showed that the vast majority of the probes did not recognize their corresponding homologous chromosomes. Alphoid sequences belonging to the suprachromosomal family 1 (chromosomes 1, 3, 5, 6, 7, 10, 12, 16, and 19) yielded very heterogeneous results: some probes gave intense signals, but always on nonhomologous chromosomes; others did not produce any hybridization signal. Almost all probes belonging to the suprachromosomal family 2 (chromosomes 2, 4, 8, 9, 13, 14, 15, 18, 20, 21, and 22) recognized a single chromosome: chromosome 11 (phylogenetic IX) in PTR and PPA and chromosome 19 (phylogenetic V) in GGO. Localization of probes of suprachromosomal family 3 (chromosomes 1, 11, 17, and X) was found to be substantially conserved in PTR and PPA, but not in GGO. Probe pDMX1, specific for the human X chromosome, was the only sequence detecting its corresponding chromosome in all three species. PPA chromosomes I, IIp, IIq, IV, V, VI, and XVIII were never labeled, even under low-stringency hybridization conditions, by the 27 alphoid probes used in this study. These results, with particular reference to differences found in the two related species PTR and PPA, suggest that alphoid centromeric sequences underwent a very rapid evolution.

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.

A panel of subchromosomal painting libraries representing over 300 regions of the human genome.

DNA samples from about 100 human-hamster somatic cell hybrids, previously characterized by conventional banding techniques, were amplified with dual-Alu PCR. The products were then used as probes in FISH experiments on normal human metaphases for an accurate cytogenetic characterization of the human material retained in each hybrid. In addition to entire chromosomes, most hybrids were found to contain one or a few chromosome fragments, as a result of rearrangements that had occurred in vitro. Forty additional primary hybrids, in which conventional cytogenetic analysis failed to reveal any complete human chromosome, contained many human chromosome fragments. More than 300 chromosome fragments were scored and their precise chromosomal location recorded. We show data indicating that subchromosomal painting libraries generated from these hybrids can be favorably used in the fine characterization of chromosomal rearrangements encountered in clinical cytogenetics or in tumor cytogenetics, and in tracking chromosomal changes that occurred in primate evolution.

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.

Upregulation of the SOX5 by promoter swapping with the P2RY8 gene in primary splenic follicular lymphoma

Upregulation of the SOX5 by promoter swapping with the P2RY8 gene in primary splenic follicular lymphoma

CBFA2T2 and C20orf112 : two novel fusion partners of RUNX1 in acute myeloid leukemia

RUNX1 (Runt-related transcription factor 1) gene, also known as AML1, maps at 21q22.3 and encodes a transcription factor crucial for normal hematopoiesis. It is frequently involved in gene fusions resulting from 35 different translocations1 (http://cgap.nci.nih.gov/Chromosomes/Mitelman). The fusion transcript 5′RUNX1/3′CBFA2T1 (alias MTG8 or ETO), resulting from a t(8;21)(q22;q22), is present in approximately 30% of acute myeloid leukemia M2 patients (AML-M2).2 Other frequent fusions, in adult AML, are the t(16;21)(q24;q22), occurring in AML-M1/M2 patients and generating a 5′RUNX1/3′CBFA2T3 chimera,4 and the t(3;21)(q26;q22), found in both de novo and secondary AML, fusing RUNX1 to MDS1, RPL22L1 (also known as EAP) or EVI1.1 In two AML cases showing a t(20;21) translocation, the partner gene was not identified.4, 5 We report here the characterization of two chimeric transcripts identified in AML translocation cases involving CBFA2T2 (core-binding factor, runt domain, α subunit; translocated to, 2), an ETO homologous gene on chromosome 20, and C20orf112.

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

Familial adenomatous polyposis: identification of a new frameshift mutation of the APC gene in an Italian family.

Familial Adenomatous Polyposis (FAP) is a premalignant disease of the gastrointestinal tract inherited as an autosomal dominant trait assigned to chromosome 5q21. The 15 exons of the APC gene responsible for the defect were amplified from the DNA of one FAP patient. SSCP analysis of the amplified DNA revealed a variant conformer of exon 10. The sequencing of the cloned PCR product showed a 1 base insertion at position 1370, creating a stop codon four nucleotides downstream. SSCP analysis of 20 family members and nucleotide sequencing of exon 10 in three affected members confirmed the Mendelian inheritance of the mutant allele.

Characterization of a hotspot region on chromosome 12 for amplification in ring chromosomes in atypical lipomatous tumors

Ring chromosomes are cytogenetic hallmarks of genomic amplification in several bone and soft tissue tumors, in particular atypical lipomatous tumors (ALT). In ALT, the ring chromosomes invariably contain amplified material from the central part of the long arm of chromosome 12, mainly 12q12→15, but often also segments from other chromosomes are involved. Previous studies have shown that one of the recurrent amplicons in ALT, located in 12q13.3‐14.1 and harboring the candidate target genes TSPAN31 and CDK4, often has a sharp centromeric border. To characterize this breakpoint region in more detail, 12 cases of ALT with ring chromosomes were analyzed by array comparative genomic hybridization and fluorescence in situ hybridization. In the seven cases showing a sharply delineated amplicon in 12q13.3‐14.1, the breakpoint region was further investigated by real time quantitative polymerase chain reaction and Vectorette PCR. The breakpoints clustered to a 146‐kb region containing 11 genes. Whereas there was no indication that the breakpoints gave rise to fusion genes, in silico analysis revealed that the breakpoint region was enriched for repeated elements that could be important for ring chromosome formation in ALT.