Maria Corsignano Guastadisegni

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.

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

Thrombocytopenia-absent-radius syndrome in a child showing a larger 1q21.1 deletion than the one in his healthy mother, and a significant downregulation of the commonly deleted genes

Thrombocytopenia-absent-radius (TAR) syndrome is a rare condition characterized by thrombocytopenia and bilateral absence of the radii with presence of both thumbs. The phenotype has a variable expression. A 200 kb minimal deletion at 1q21.1 is present in all patients. However, the microdeletion, ranging up to 1100 kb in length, is not sufficient to cause the disease. Indeed it is present in 75-80% of unaffected parents. It is assumed that the phenotype develops only in the presence of one or more additional, as-yet-unknown, deletion modifiers (mTARs). We report here on a child affected by TAR syndrome associated with Langerhans cell histiocytosis. Unexpectedly, he showed a 2.029 kb deletion at 1q21.1, almost twice that of the unaffected mother (957 kb). Interestingly, the mother-to-son increased size of the deleted region was already observed in two cases of constitutional diseases, although both resulting as chromosomal terminal deletions. Noteworthy, qPCR experiments, never before performed for patients with TAR syndrome, disclosed that the proband had a statistically significant downregulation of the majority of the genes mapping inside the part of the deletion shared with the mother. The mother, on the contrary, did not show the same downregulation. In summary, the present report adds new insights on the pathogenesis of TAR syndrome, that may represent fruitful directions for future research.