Involvement of DPP9 in gene fusions in serous ovarian carcinoma.

BACKGROUND: A fusion gene is a hybrid gene consisting of parts from two previously independent genes. Chromosomal rearrangements leading to gene breakage are frequent in high-grade serous ovarian carcinomas and have been reported as a common mechanism for inactivating tumor suppressor genes. However, no fusion genes have been repeatedly reported to be recurrent driver events in ovarian carcinogenesis. We combined genomic and transcriptomic information to identify novel fusion gene candidates and aberrantly expressed genes in ovarian carcinomas. METHODS: Examined were 19 previously karyotyped ovarian carcinomas (18 of the serous histotype and one undifferentiated). First, karyotypic aberrations were compared to fusion gene candidates identified by RNA sequencing (RNA-seq). In addition, we used exon-level gene expression microarrays as a screening tool to identify aberrantly expressed genes possibly involved in gene fusion events, and compared the findings to the RNA-seq data. RESULTS: We found a DPP9-PPP6R3 fusion transcript in one tumor showing a matching genomic 11;19-translocation. Another tumor had a rearrangement of DPP9 with PLIN3. Both rearrangements were associated with diminished expression of the 3' end of DPP9 corresponding to the breakpoints identified by RNA-seq. For the exon-level expression analysis, candidate fusion partner genes were ranked according to deviating expression compared to the median of the sample set. The results were collated with data obtained from the RNA-seq analysis. Several fusion candidates were identified, among them TMEM123-MMP27, ZBTB46-WFDC13, and PLXNB1-PRKAR2A, all of which led to stronger expression of the 3' genes. In view of our previous findings of nonrandom rearrangements of chromosome 19 in this cancer type, particular emphasis was given to changes of this chromosome and a DDA1-FAM129C fusion event was identified. CONCLUSIONS: We have identified novel fusion gene candidates in high-grade serous ovarian carcinoma. DPP9 was involved in two different fusion transcripts that both resulted in deregulated expression of the 3' end of the transcript and thus possible loss of the active domains in the DPP9 protein. The identified rearrangements might play a role in tumorigenesis or tumor progression.

Low frequency of ESRRA-C11orf20 fusion gene in ovarian carcinomas.

The identification of recurrent gene fusions in common epithelial cancers--for example, TMPRSS2/ERG in prostate cancer and EML4/ALK in nonsmall cell lung carcinomas--has raised the question of whether fusion genes are pathogenetically important also in ovarian carcinomas. The first recurrent fusion transcript in serous ovarian carcinomas was reported by Salzman et al. in 2011, who used deep paired-end sequencing to detect the fusion gene ESRRA-C11orf20 in 10 out of 67 (15%) serous ovarian carcinomas examined, a finding that holds great promise for our understanding of ovarian tumorigenesis as well as, potentially, for new treatment strategies. We wanted to test how frequent the ESRRA/C11orf20 fusion is in ovarian carcinomas of all subtypes, and therefore examined a series of 230 ovarian carcinomas of which 197 were of the serous subtype and 163 of the 197 were of stages III and IV--that is, the very same carcinoma subset where the fusion transcript had been found. We performed PCR and high-throughput sequencing analyses in search of the fusion transcript. We used the same primers described previously for the detection of the fusion and the same primer combination, but found no ESRRA/C11orf20 fusion in our series. A synthetic DNA plasmid containing the reported ESRRA/C11orf20 fusion was included as a positive control for our PCR experiments. Data from high-throughput sequencing of 23 ovarian carcinomas were screened in search of alternative partner(s) for the ESRRA and/or C11orf20 gene, but none was found. We conclude that the frequency of the ESRRA/C11orf20 gene fusion in serous ovarian carcinomas of stages III and IV must be considerable less than that reported previously (0/163 in our experience compared with 10/67 in the previous study). At the very least, it seems clear that the said fusion cannot be a common pathogenetic event in this tumor type.

The genome sequence of Atlantic cod reveals a unique immune system.

Atlantic cod (Gadus morhua) is a large, cold-adapted teleost that sustains long-standing commercial fisheries and incipient aquaculture. Here we present the genome sequence of Atlantic cod, showing evidence for complex thermal adaptations in its haemoglobin gene cluster and an unusual immune architecture compared to other sequenced vertebrates. The genome assembly was obtained exclusively by 454 sequencing of shotgun and paired-end libraries, and automated annotation identified 22,154 genes. The major histocompatibility complex (MHC) II is a conserved feature of the adaptive immune system of jawed vertebrates, but we show that Atlantic cod has lost the genes for MHC II, CD4 and invariant chain (Ii) that are essential for the function of this pathway. Nevertheless, Atlantic cod is not exceptionally susceptible to disease under natural conditions. We find a highly expanded number of MHC I genes and a unique composition of its Toll-like receptor (TLR) families. This indicates how the Atlantic cod immune system has evolved compensatory mechanisms in both adaptive and innate immunity in the absence of MHC II. These observations affect fundamental assumptions about the evolution of the adaptive immune system and its components in vertebrates.

RNA sequencing identifies fusion of the EWSR1 and YY1 genes in mesothelioma with t(14;22)(q32;q12).

Mesothelioma is a rare but very aggressive tumor derived from mesothelial cells. A number of often complex but nonrandom cytogenetic abnormalities have been found in these tumors, resulting in loss of chromosome bands 14q32 and 22q12 in more than 35% of the cases. In this study, we used RNA sequencing to search for fusion transcripts in a mesothelioma carrying a t(14;22)(q32;q12) as the sole chromosomal aberration and found an EWSR1-YY1 and its reciprocal YY1-EWSR1 fusion transcript. Screening 15 additional cases of mesothelioma from which we had RNA but no cytogenetic information, we identified one more tumor carrying an EWSR1-YY1 fusion gene but not the reciprocal YY1-EWSR1 transcript. RT-polymerase chain reaction and sequencing showed that in both cases exon 8 of EWSR1 (nucleotide 1,139, accession number NM_013986 version 3, former exon 7 in sequence with accession number X66899) was fused to exon 2 of YY1 (nucleotide 1,160, accession number NM_003403 version 3). The EWSR1 breakpoint in exon 8 in the EWSR1-YY1 chimeric transcript is similar to what is found in other fusions involving EWSR1 such as EWSR1-FLI1, EWSR1-DDIT3, and EWSR1-ATF1. The EWSR1-YY1-encoded protein is an abnormal transcription factor with the transactivation domain of EWSR1 and the DNA-binding domain of YY1. This is the first study to detect a specific fusion gene in mesothelioma (the reason how frequent the EWSR1-YY1 fusion is remains uncertain) and also the first time that direct involvement of YY1 in oncogenesis has been demonstrated.

Fusion of the ZC3H7B and BCOR genes in endometrial stromal sarcomas carrying an X;22-translocation.

Endometrial stromal sarcomas (ESS) are genetically heterogeneous uterine tumors in which a JAZF1-SUZ12 chimeric gene resulting from the chromosomal translocation t(7;17)(p15;q21) as well as PHF1 rearrangements (in chromosomal band 6p21) with formation of JAZF1-PHF1, EPC1-PHF1, and MEAF6-PHF1 chimeras have been described. Here, we investigated two ESS characterized cytogenetically by the presence of a der(22)t(X;22)(p11;q13). Whole transcriptome sequencing one of the tumors identified a ZC3H7-BCOR chimeric transcript. Reverse transciptase-PCR with the ZC3H7B forward and BCOR reverse primer combinations confirmed the presence of a ZC3H7-BCOR chimeric transcript in both ESS carrying a der(22)t(X;22) but not in a control ESS with t(1;6) and the MEAF6-PHF1 fusion. Sequencing of the amplified cDNA fragments showed that in both cases ESS exon 10 of ZC3H7B (from 22q13; accession number NM_017590 version 4) was fused to exon 8 of BCOR (from Xp11; accession number NM_001123385 version 1). Reciprocal multiple BCOR-ZC3H7B cDNA fragments were amplified in only one case suggesting that ZC3H7B-BCOR, on the der(22)t(X;22), is the pathogenetically important fusion gene. The putative ZC3H7B-BCOR protein would contain the tetratricopeptide repeats and LD motif from ZC3H7B and the AF9 binding site (1093-1233aa), the 3 ankyrin repeats (1410-1509 aa), and the NSPC1 binding site of BCOR. Although the presence of these motifs suggests various functions of the chimeric protein, it is possible that its most important role may be in epigenetic regulation. Whether or not the (patho)genetic subsets JAZF1-SUZ12, PHF1 rearrangements, and ZC3H7B-BCOR correspond to any phenotypic, let alone clinically important, differences in ESS remain unknown.

Intestinal fatty acid binding protein (fabp2) in Atlantic salmon (Salmo salar): Localization and alteration of expression during development of diet induced enteritis.

In the present study full-length cDNAs corresponding to three isoforms of intestinal fatty acid binding protein (fabp2) in Atlantic salmon were cloned and characterized. Gene expression of fabp2 was observed in all tissues investigated, although differences were observed between isoforms. The highest fabp2a1, fabp2a2, and fabp2b expression was in the intestine. A 15kDa protein, corresponding to putative Fabp2 protein, was identified by immunoblotting using anti-human Fabp2 antibody. Immunoblotting and immunohistochemistry confirmed that Fabp2 protein was present in most Atlantic salmon tissues. Similar to gene expression, intestinal tissues had the highest Fabp2 protein levels, decreasing gradually from proximal to distal intestine. During development of distal intestinal inflammation caused by dietary soybean meal from 0 to 21days, Fabp2 decreased significantly on both transcriptional and protein levels. The reduction in Fabp2 was preceded by a down regulation of peroxisome proliferator activated receptor (ppar) alpha and gamma, fabp2's presumed regulatory proteins, and followed by a progressive increase in proliferating cell nuclear antigen (Pcna) staining. Results illustrate that the early decline of distal intestinal fabp2 was likely caused by a down regulation of their regulatory proteins, but at later time points reduced Fabp2 may largely be due to a less mature enterocyte population resulting from rapid cell turnover.

Pigment-producing granulomatous myopathy in Atlantic salmon: a novel inflammatory response.

Melanin comprises a complex group of pigmented polymers whose primary function is ascribed to dermal solar protection, but may also have an interesting role in innate immunity. In ectothermic vertebrates, melanogenesis is reported in leukocyte populations, but it is not known if this occurs in connection with inflammatory reactions. Melanin accumulations in ectopic locations, in particular muscle, represent a serious quality problem in salmon production. Here, we investigated such changes for the expression of dopachrome tautomerase and tyrosinase as well as some important immune genes and pathogens. Furthermore, the nature of the pathological changes was addressed by morphological methods. Gene transcripts encoding key enzymes in melanogenesis, suggesting a de novo melanin synthesis in pigmented muscle, were found. MHC class II transcripts were up-regulated and there was no indication of bacterial or viral infection. The histological examination revealed granulomatous inflammation with distribution of MHC class II positive cells and T cells, analogous to the pattern found in mammals. Importantly, in contrast to mammals pigmented cells were contributing in the inflammation. We demonstrate that melanin production occurs in granulomatous inflammation in salmon, revealing a close and hitherto unreported link between the pigmentary and immune systems.

Regulated expression of a transgene introduced on an oriP/EBNA-1 PAC shuttle vector into human cells.

BACKGROUND: Sequencing of the human genome has led to most genes being available in BAC or PAC vectors. However, limited functional information has been assigned to most of these genes. Techniques for the manipulation and transfer of complete functional units on large DNA fragments into human cells are crucial for the analysis of complete genes in their natural genomic context. One limitation of the functional studies using these vectors is the low transfection frequency. RESULTS: We have constructed a shuttle vector, pPAC7, which contains both the EBNA-1 gene and oriP from the Epstein-Barr virus allowing stable maintenance of PAC clones in the nucleus of human cells. The pPAC7 vector also contains the EGFP reporter gene, which allows direct monitoring of the presence of PAC constructs in transfected cells, and the Bsr-cassette that allows highly efficient and rapid selection in mammalian cells by use of blasticidin. Positive selection for recombinant PAC clones is obtained in pPAC7 because the cloning sites are located within the SacBII gene. We show regulated expression of the CDH3 gene carried as a 132 kb genomic insert cloned into pPAC7, demonstrating that the pPAC7 vector can be used for functional studies of genes in their natural genomic context. Furthermore, the results from the transfection of a range of pPAC7 based constructs into two human cell lines suggest that the transfection efficiencies are not only dependent on construct size. CONCLUSION: The shuttle vector pPAC7 can be used to transfer large genomic constructs into human cells. The genes transferred could potentially contain all long-range regulatory elements, including their endogenous regulatory promoters. Introduction of complete genes in PACs into human cells would potentially allow complementation assays to identify or verify the function of genes affecting cellular phenotypes.

Characterization of a BAC library from channel catfish Ictalurus punctatus: indications of high levels of chromosomal reshuffling among teleost genomes.

The CHORI-212 bacterial artificial chromosome (BAC) library was constructed by cloning EcoRI/EcoRI partially digested DNA into the pTARBAC2.1 vector. The library has an average insert size of 161 kb, and provides 10.6-fold coverage of the channel catfish haploid genome. Screening of 32 genes using overgo or cDNA probes indicated that this library had a good representation of the genome as all tested genes existed in the library. We previously reported sequencing of approximately 25,000 BAC ends that generated 20,366 high-quality BAC end sequences (BES) and identified a large number of sequences similar to known genes using BLASTX searches. In this work, particular attention was given to identification of BAC mate pairs with known genes from both ends. When identified, comparative genome analysis was conducted to determine syntenic regions of the catfish genome with the genomes of zebrafish and Tetraodon. Of the 141 mate pairs with known genes from channel catfish, conserved syntenies were identified in 34 (24.1%), with 30 conserved in the zebrafish genome and 14 conserved in the Tetraodon genome. Additional analysis of three of the 34 conserved syntenic groups by direct sequencing indicated conserved gene contents in all three species. This indicates that comparative genome analysis may provide shortcuts to genome analysis in catfish, especially for short genomic regions once the conserved syntenies are identified.

A highly redundant BAC library of Atlantic salmon (Salmo salar): an important tool for salmon projects.

BACKGROUND: As farming of Atlantic salmon is growing as an aquaculture enterprise, the need to identify the genomic mechanisms for specific traits is becoming more important in breeding and management of the animal. Traits of importance might be related to growth, disease resistance, food conversion efficiency, color or taste. To identify genomic regions responsible for specific traits, genomic large insert libraries have previously proven to be of crucial importance. These large insert libraries can be screened using gene or genetic markers in order to identify and map regions of interest. Furthermore, large-scale mapping can utilize highly redundant libraries in genome projects, and hence provide valuable data on the genome structure. RESULTS: Here we report the construction and characterization of a highly redundant bacterial artificial chromosome (BAC) library constructed from a Norwegian aquaculture strain male of Atlantic salmon (Salmo salar). The library consists of a total number of 305,557 clones, in which approximately 299,000 are recombinants. The average insert size of the library is 188 kbp, representing 18-fold genome coverage. High-density filters each consisting of 18,432 clones spotted in duplicates have been produced for hybridization screening, and are publicly available 1. To characterize the library, 15 expressed sequence tags (ESTs) derived overgos and 12 oligo sequences derived from microsatellite markers were used in hybridization screening of the complete BAC library. Secondary hybridizations with individual probes were performed for the clones detected. The BACs positive for the EST probes were fingerprinted and mapped into contigs, yielding an average of 3 contigs for each probe. Clones identified using genomic probes were PCR verified using microsatellite specific primers. CONCLUSION: Identification of genes and genomic regions of interest is greatly aided by the availability of the CHORI-214 Atlantic salmon BAC library. We have demonstrated the library's ability to identify specific genes and genetic markers using hybridization, PCR and fingerprinting experiments. In addition, multiple fingerprinting contigs indicated a pseudo-tetraploidity of the Atlantic salmon genome. The highly redundant CHORI-214 BAC library is expected to be an important resource for mapping and sequencing of the Atlantic salmon genome.

Fusion genes with ALK as recurrent partner in ependymoma-like gliomas: a new brain tumor entity?

BACKGROUND: We have previously characterized 19 ependymal tumors using Giemsa banding and high-resolution comparative genomic hybridization. The aim of this study was to analyze these tumors searching for fusion genes. METHODS: RNA sequencing was performed in 12 samples. Potential fusion transcripts were assessed by seed count and structural chromosomal aberrations. Transcripts of interest were validated using fluorescence in situ hybridization and PCR followed by direct sequencing. RESULTS: RNA sequencing identified rearrangements of the anaplastic lymphoma kinase gene (ALK) in 2 samples. Both tumors harbored structural aberrations involving the ALK locus 2p23. Tumor 1 had an unbalanced t(2;14)(p23;q22) translocation which led to the fusion gene KTN1-ALK. Tumor 2 had an interstitial del(2)(p16p23) deletion causing the fusion of CCDC88A and ALK. In both samples, the breakpoint of ALK was located between exons 19 and 20. Both patients were infants and both tumors were supratentorial. The tumors were well demarcated from surrounding tissue and had both ependymal and astrocytic features but were diagnosed and treated as ependymomas. CONCLUSIONS: By combining karyotyping and RNA sequencing, we identified the 2 first ever reported ALK rearrangements in CNS tumors. Such rearrangements may represent the hallmark of a new entity of pediatric glioma characterized by both ependymal and astrocytic features. Our findings are of particular importance because crizotinib, a selective ALK inhibitor, has demonstrated effect in patients with lung cancer harboring ALK rearrangements. Thus, ALK emerges as an interesting therapeutic target in patients with ependymal tumors carrying ALK fusions.

Sequential combination of karyotyping and RNA-sequencing in the search for cancer-specific fusion genes.

Cancer-specific fusion genes are often caused by cytogenetically visible chromosomal rearrangements such as translocations, inversions, deletions or insertions, they can be the targets of molecular therapy, they play a key role in the accurate diagnosis and classification of neoplasms, and they are of prognostic impact. The identification of novel fusion genes in various neoplasms therefore not only has obvious research importance, but is also potentially of major clinical significance. The "traditional" methodology to detect them began with cytogenetic analysis to find the chromosomal rearrangement, followed by utilization of fluorescence in situ hybridization techniques to find the probe which spans the chromosomal breakpoint, and finally molecular cloning to localize the breakpoint more precisely and identify the genes fused by the chromosomal rearrangement. Although laborious, the above-mentioned sequential approach is robust and reliable and a number of fusion genes have been cloned by such means. Next generation sequencing (NGS), mainly RNA sequencing (RNA-Seq), has opened up new possibilities to detect fusion genes even when cytogenetic aberrations are cryptic or information about them is unknown. However, NGS suffers from the shortcoming of identifying as "fusion genes" also many technical, biological and, perhaps in particular, clinical "false positives," thus making the assessment of which fusions are important and which are noise extremely difficult. The best way to overcome this risk of information overflow is, whenever reliable cytogenetic information is at hand, to compare karyotyping and sequencing data and concentrate exclusively on those suggested fusion genes that are found in chromosomal breakpoints. This article is part of a Directed Issue entitled: Rare Cancers.

Fusion of ZMYND8 and RELA genes in acute erythroid leukemia.

Acute erythroid leukemia was diagnosed in a 4-month-old boy. Cytogenetic analysis of bone marrow (BM) cells showed a t(11;20)(p11;q11) translocation. RNA extracted from the BM was sequenced and analyzed for fusion transcripts using the software FusionMap. A ZMYND8-RELA fusion was ranked first. RT-PCR and direct sequencing verified the presence of an in frame ZMYND8-RELA chimeric transcript. Fluorescence in situ hybridization showed that the ZMYND8-RELA was located on the p12 band of der(11); therefore a cytogenetically invisible pericentric inversion in chromosome 11 must have taken place besides the translocation. The putative ZMYND8-RELA fusion protein contains the Zinc-PHD finger domain, a bromodomain, a PWWP domain, a MYND type of zinc finger of ZMYND8, and the entire RELA protein, indicating that it might act leukemogenically by influencing several cellular processes including the NF-kappa-B pathway.

A novel TCF3-HLF fusion transcript in acute lymphoblastic leukemia with a t(17;19)(q22;p13).

A 10-year-old boy was admitted to the hospital because of anemia detected after a two week history of fatigue, dizziness, nausea, headaches, and weight loss. A bone marrow investigation confirmed a diagnosis of acute lymphoblastic leukemia of the B-cell precursor phenotype. Chromosome G-banding analysis yielded the karyotype 46,XY,t(17;19)(q22;p13), and fluorescence in situ hybridization (FISH) analysis showed rearrangement of the genes TCF3 (on 19p13; accession number NM_03200 version 3) and HLF (on 17q22; accession number NM_002126 version 4) with the generation of a TCF3-HLF chimera. Polymerase chain reaction and sequencing analyses demonstrated the presence of two in-frame chimeric TCF3-HLF transcripts. In the first one, which corresponds to a type 2 fusion, exon 15 of TCF3 is fused to exon 4 of HLF. In the second, described here for the first time and named type 3, exon 14 of TCF3 is fused to exon 4 of HLF. Whether the type 3 chimeric transcript has the same DNA binding and transcriptional regulatory effect as type 1 and type 2 TCF3-HLF chimeras remains to be seen.

Myelodysplastic syndrome with a t(2;11)(p21;q23-24) and translocation breakpoint close to miR-125b-1.

The upregulation of oncogenes and the formation of fusion genes are commonly observed in hematological malignancies with recurring balanced translocations. However, in some malignancies exhibiting balanced chromosomal rearrangements, neither oncogene deregulation nor generation of fusion genes appears to be involved, suggesting that other mechanisms are at play. In the rare myelodysplastic syndrome (MDS) containing a t(2;11)(p21;q23-24) translocation, breakpoints near a microRNA locus, miR-125b-1, in 11q24 have been suggested to be pathogenetically involved. Here we report the detailed mapping and sequencing of the breakpoint located only 2 kilobases from miR-125b-1 in an MDS patient with a t(2;11)(p21;q23-24).

Novel fusion of MYST/Esa1-associated factor 6 and PHF1 in endometrial stromal sarcoma.

Rearrangement of chromosome band 6p21 is recurrent in endometrial stromal sarcoma (ESS) and targets the PHF1 gene. So far, PHF1 was found to be the 3' partner in the JAZF1-PHF1 and EPC1-PHF1 chimeras but since the 6p21 rearrangements involve also other chromosomal translocation partners, other PHF1-fusions seem likely. Here, we show that PHF1 is recombined with a novel fusion partner, MEAF6 from 1p34, in an ESS carrying a t(1;6)(p34;p21) translocation as the sole karyotypic anomaly. 5'-RACE, RT-PCR, and sequencing showed the presence of an MEAF6-PHF1 chimera in the tumor with exon 5 of MEAF6 being fused in-frame to exon 2 of PHF1 so that the entire PHF1 coding region becomes the 3' terminal part of the MEAF6-PHF1 fusion. The predicted fusion protein is composed of 750 amino acids and contains the histone acetyltransferase subunit NuA4 domain of MEAF6 and the tudor, PHD zinc finger, and MTF2 domains of PHF1. Although the specific functions of the MEAF6 and PHF1 proteins and why they are targeted by a neoplasia-specific gene fusion are not directly apparent, it seems that rearrangement of genes involved in acetylation (EPC1, MEAF6) and methylation (PHF1), resulting in aberrant gene expression, is a common theme in ESS pathogenesis.

Whole-transcriptome sequencing identifies novel IRF2BP2-CDX1 fusion gene brought about by translocation t(1;5)(q42;q32) in mesenchymal chondrosarcoma.

Mesenchymal chondrosarcomas (MCs) account for 3-10% of primary chondrosarcomas. The cytogenetic literature includes only ten such tumours with karyotypic information and no specific aberrations have been identified. Using a purely molecular genetic approach a HEY1-NCOA2 fusion gene was recently detected in 10 of 15 investigated MCs. The fusion probably arises through intrachromosomal rearrangement of chromosome arm 8 q. We report a new case of MC showing a t(1;5)(q42;q32) as the sole karyotypic aberration. Through FISH and whole transcriptome sequencing analysis we found a novel fusion between the IRF2BP2 gene and the transcription factor CDX1 gene arising from the translocation. The IRF2BP2-CDX1 has not formerly been described in human neoplasia. In our hospital's archives three more cases of MC were found, and we examined them looking for the supposedly more common HEY1-NCOA2 fusion, finding it in all three tumours but not in the case showing t(1;5) and IRF2BP2-CDX1 gene fusion. This demonstrates that genetic heterogeneity exists in mesenchymal chondrosarcoma.

Identification of chromosomal breakpoints of cancer-specific translocations by rolling circle amplification and long-distance inverse PCR.

We describe the use of rolling circle amplification and long-distance inverse polymerase chain reaction (LD-PCR) to identify chromosomal breakpoints and fusion genes in cancer cells carrying acquired translocations. This approach produced enough template for 100 inverse PCR reaction from as little as 20 ng of patient DNA, consequently enabling the use of up to 500 times less patient DNA compared to standard inverse PCR. The method is based on identifying restriction sites in a putative breakpoint area in a cancer-specific translocation, followed by circularization and amplification of the restriction DNA products by using T4 DNA ligase and Phi29 enzyme, respectively. The amplified DNA thus obtained is used as a template in long-distance inverse PCR to amplify and detect the precise breakpoint of the chromosomal rearrangements in question by sequencing of the obtained PCR products. We demonstrate the feasibility of this approach by identifying fusion genes TAF15-ZNF384 (brought about by a (12;17)(p13;q21) translocation) and BCR-ABL1 (produced by a (9:22)(q34;q11.2) translocation) in five leukemia samples. The application of rolling circle amplification before inverse PCR may be particularly useful in the search for chromosomal breakpoints and fusion genes brought about by new translocations when only minute amounts of DNA are available from the sampled malignant lesion.

Identification of the TAF15-ZNF384 fusion gene in two new cases of acute lymphoblastic leukemia with a t(12;17)(p13;q12).

We report the clinical, cytogenetic, and molecular data of two patients diagnosed with acute lymphoblastic leukemia characterized by the rare translocation t(12;17)(p13;q12). This translocation has been reported in 25 cases and its putative molecular consequence, the formation of a TAF15-ZNF384 fusion gene, in only six cases. We used fluorescence in situ hybridization followed by long-range polymerase chain reaction to find the translocation breakpoints. A fusion between TAF15 and ZNF384 was identified and confirmed by nucleotide sequencing. Our results confirm that the t(12;17)(p13;q12) leading to a TAF15-ZNF384 fusion gene characterizes a specific subgroup of acute lymphoblastic leukemia and suggest that two different breakpoints in TAF15 may be involved. Whether the two variants of the TAF15-ZNF384 fusion that these correspond to are in any way hematologically or prognostically different, is unknown.