Aggressive systemic mastocytosis with the co-occurrence of PRKG2::PDGFRB, KAT6A::NCOA2, and RXRA::NOTCH1 fusion transcripts and a heterozygous RUNX1 frameshift mutation.

Systemic mastocytosis (SM) is a myeloproliferative neoplasm displaying abnormal mast cell proliferation. It is subdivided into different forms, including aggressive systemic mastocytosis (ASM) and systemic mastocytosis with an associated hematologic neoplasm (SM-AHN). Oncogenic genetic alterations include point mutations, mainly the KIT D816V, conferring poor prognosis and therapy resistance, and fusion genes, with those involving PDGFRA/PDGFRB as the most recurrent events. We here describe an ASM case negative to the KIT D816V and JAK2 V617F alterations but showing a RUNX1 frameshift heterozygous mutation and the co-occurrence of three fusion transcripts. The first one, PRKG2::PDGFRB, was generated by a balanced t(4;5)(q24;q32) translocation as the sole abnormality. Other two novel chimeras, KAT6A::NCOA2 and RXRA::NOTCH1, originated from cryptic intra-chromosomal abnormalities. The patient rapidly evolved towards SM-AHN, characterized by the persistence of the PRKG2::PDGFRB chimera, due to the presence of an extra copy of the der(5)t(4;5)(q24;q34) chromosome and an increase in the RUNX1 mutation allelic frequency. The results indicated that the transcriptional landscape and the mutational profile of SM deserve attention to predict the evolution and prognosis of this complex disease, whose classification criteria are still a matter of debate.

Concurrent chromothripsis events in a case of TP53 depleted acute myeloid leukemia with myelodysplasia-related changes.

Acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) is a heterogeneous hematological disorder defined by morphological, genetic, and clinical features. Patients with AML-MRC often show cytogenetic changes, which are associated with poor prognosis. Straightforward criteria for AML-MRC diagnosis and a more rigorous characterization of the genetic abnormalities accompanying this disease are needed. Here we describe an informative AML-MRC case, showing two separate, but concurrent, chromothripsis events, occurred at the onset of the tumor, and originating an unbalanced t(5;7) translocation and a derivative chromosome 12 with a highly rearranged short arm. Conversely, despite chromothripsis has been often associated with genomic amplification in cancer, in this case a large marker chromosome harboring amplified sequences from chromosomes 19 and 22 arose from a stepwise mechanism. Notably, the patient also showed a TP53 mutated status, known to be associated with an increased susceptibility towards chromothripsis and a poor prognosis. Our results indicate that multiple chromothripsis events may occur early in neoplastic transformation and act in a synergistic way with progressive chromosomal alterations to determine a dramatic impact on disease outcome, as suggested by the gene expression profile analysis.

A novel fusion 5'AFF3/3'BCL2 originated from a t(2;18)(q11.2;q21.33) translocation in follicular lymphoma.

Follicular lymphoma is the second most frequent type of non-Hodgkin's lymphoma in adults. The basic molecular defect consists of the t(14;18)(q32;q21) translocation, juxtaposing the B-cell lymphoma protein 2 gene BCL2 to the immunoglobulin heavy chain locus IGH@, and leading to the antiapoptotic BCL2 protein overproduction. Variations in the t(14;18) are rare and can be classified into two categories: (i) simple variants, involving chromosomes 18 and 2, or 22, in which the fusion partner of BCL2 is the light-chain IGK@ or IGL@; (ii) complex variant translocations occurring among chromosomes 14, 18 and other chromosomes. We report a follicular lymphoma case showing BCL2 overexpression, detected by immunohistochemistry and real-time quantitative PCR, consequently to the formation of a novel fusion gene between the 5' of the lymphoid nuclear transcriptional activator gene AFF3 at 2q11.2, and the 3' of BCL2. This case shows evidence, for the first time, of BCL2 overexpression consequently to the fusion of BCL2 to a non-IG partner locus.

Identification of a novel amplicon at distal 17q containing the BIRC5/SURVIVIN gene in malignant peripheral nerve sheath tumours.

Previous studies have suggested that amplification of genes, notably the TOP2A gene, on chromosome arm 17q may be important for the development of malignant peripheral nerve sheath tumour (MPNST). In order to study the frequency, distribution, and chromosomal organization of rearrangements at 17q, interphase and metaphase fluorescence in situ hybridization (FISH) were used to evaluate copy number changes at 17q in 28 MPNSTs. Increased copy numbers were seen for the ERBB2 and TOP2A genes in eight and nine cases, respectively, supporting a potential role for these two genes in MPNST tumourigenesis. Net gain of distal 17q material was observed in 16 of the 28 MPNSTs, with high-level gain in three cases, and was associated with poor outcome. Among the 26 patients for whom follow-up data were available, gain of distal 17q was present in 11 of 12 tumours that had metastasized, compared with 4 of 14 of those that had not metastasized. Detailed FISH mapping analysis of metaphase spreads identified a 2 Mb commonly gained/amplified region at 17q25. Among the genes mapping to this region, BIRC5, which encodes the baculoviral IAP repeat-containing protein 5/survivin protein, is a strong candidate target gene for amplification, as it has been previously shown to be overexpressed in neurofibromatosis type 1-associated MPNST. Three other genes that co-amplified with BIRC5 represent other potential candidate genes: PTDSR involved in apoptosis; SEPT9 overexpressed in human malignant brain tumours; and SOCS3 involved in cell survival and differentiation of neurons.

Novel rearrangement of chromosome band 22q11.2 causing 22q11 microdeletion syndrome-like phenotype and rhabdoid tumor of the kidney.

The 22q11.2 microdeletion syndrome is the most frequent microdeletion syndrome in humans, yet its genetic basis is complex and is still not fully understood. Most patients harbor a 3-Mb deletion (typically deleted region [TDR]), but occasionally patients with atypical deletions, some of which do not overlap with each other and/or the TDR, have been described. Microduplication of the TDR leads to a phenotype similar, albeit not identical, to the deletion of this region. Here we present a child initially suspected of having 22q11 microdeletion syndrome, who in addition developed a fatal malignant rhabdoid tumor of the kidney. Detailed cytogenetic and molecular analyses revealed a complex de novo rearrangement of band q11 of the paternally derived chromosome 22. This aberration exhibited two novel features. First, a microduplication of the 22q11 TDR was associated with an atypical 22q11 microdeletion immediately telomeric of the duplicated region. Second, this deletion was considerably larger than previously reported atypical 22q11 deletions, spanning 2.8 Mb and extending beyond the SMARCB1/SNF5/INI1 tumor suppressor gene, whose second allele harbored a somatic frameshift-causing sequence alteration in the patient's tumor. Two nonallelic homologous recombination events between low-copy repeats (LCRs) could explain the emergence of this novel and complex mutation associated with the phenotype of 22q11 microdeletion syndrome.

A novel chromosomal translocation t(3;7)(q26;q21) in myeloid leukemia resulting in overexpression of EVI1.

The EVI1 proto-oncogene encodes a nuclear zinc finger protein that acts as a transcription repressor factor. In myeloid leukemia it is often activated by chromosomal rearrangements involving band 3q26, where the gene has been mapped. Here we report two leukemia cases [a chronic myeloid leukemia blast crisis (CML-BC) and an acute myeloid leukemia (AML) M4] showing a t(3;7)(q26;q21) translocation in a balanced and unbalanced form, respectively. Fluorescent in situ hybridization (FISH) analysis revealed that both patients showed a breakpoint on chromosome 3 inside the clone RP11-33A1 containing the EVI1 oncogene and, on chromosome 7, inside the clone RP11-322M5, partially containing the CDK6 oncogene which is a D cyclin-dependent kinase gene, observed to be overexpressed and disrupted in many hematological malignancies. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed overexpression of EVI1 in both cases, but excluded the presence of any CDK6/ EVI1 fusion transcript. CDK6 expression was also detected. Together, these data indicate that EVI1 activation is likely due not to the generation of a novel fusion gene with CDK6 but to a position effect dysregulating its transcriptional pattern.

Deletions on der(9) chromosome in adult Ph-positive acute lymphoblastic leukemia occur with a frequency similar to that observed in chronic myeloid leukemia.

The t(9;22)(q34;q11), generating the Philadelphia chromosome (Ph), is found in more than 90% of patients with chronic myeloid leukemia (CML) and in 15-30% of adults with acute lymphoblastic leukemia (ALL). Different groups have recently described the presence of large genomic deletions adjacent to the translocation breakpoint on the derivative chromosome 9 in 9-16% of CML patients. In the present paper, we report a FISH study of 45 Ph+ adult ALL patients with the aim of investigating the presence of deletions on derivative chromosome 9. In four (9%) of 45 cases, all showing an M-bcr, we detected deletions on der(9). The frequency of deletions we observed is similar to that reported in CML patients. The association of an M-bcr breakpoint and deletions appears significant (P=0.03). Some authors have suggested a very low incidence of der(9) deletions in ALL. This discrepancy can be explained by taking into account the low percentage of M-bcr ALL patients in the latter study (18%) compared to the present one (44%).

Alternative promoters drive the expression of the gene encoding the mouse axonal glycoprotein F3/contactin.

F3/Contactin is a neuronal glycoprotein which mediates axonal growth control via complex interactions with a number of cell surface or matrix components. As part of this developmental role, its expression undergoes differential regulation during the maturation of definite neuronal populations within the central and peripheral nervous tissue. To elucidate the underlying molecular mechanisms we study here the organization of the regulatory region of the mouse F3/Contactin gene. We show that this region displays peculiar features in that it spans more than 80 kb, bears very large introns and includes four untranslated exons which undergo complex splicing events leading to 11 potential arrangements of the F3/Contactin mRNA 5' end. Within this region we identify three alternative neurospecific promoters which, as deduced from the developmental profile of the associated 5' exons (A1,C1,0), drive two different patterns of F3/Contactin gene expression. The activity of the A1 exon-associated promoter displays only minor developmental changes and is likely to contribute to the basal level of the F3/Contactin gene expression; by contrast, the activities of the exon C1- and exon 0-associated promoters are significantly upregulated at the end of the first postnatal week. The data indicate that differential regulation of the F3/Contactin expression during development may depend upon alternative utilization of distinct promoter elements and may involve complex splicing events of the 5' untranslated exons. Several consensuses for homeogene transcription factors are scattered within the identified regulatory region, in agreement with the general assumption of homeotic gene regulation of neural morphoregulatory molecules.

Acute promyelocytic leukemia with additional chromosome abnormalities in a renal transplant case.

Some cases of acute myeloid leukemia following organ transplant (PT-AML) have been published in the literature. We report the second case of acute promyelocytic leukemia (APL), which developed post-transplant and immunosuppressive treatment, in a 50-year-old male who had undergone a renal transplant. At diagnosis he presented typical t(15;17)(q12;q13) with additional abnormalities, including +8,t(13;22)(q12;q13) and an abnormal chromosome 1 which was better characterized by fluorescence in situ hybridization (FISH). He obtained cytological, karyotypic and molecular complete remission (CR) with induction treatment according to the all-trans retinoic acid + idarubican (AIDA) protocol; after 12 months, he relapsed (molecular relapse) and achieved molecular remission with all-trans retinoic acid (ATRA) plus mitoxantrone and cytosine arabinoside. After a further 14 months, he was treated with arsenic trioxide for cytological relapse and obtained a third CR; at the cytological relapse the karyotype showed 47,XY,+8, t(15;17)(q22;q21),t(13;22)(q12;q13),der(22)t(1;22)(p22;q13). He is alive 3.3 years after diagnosis of APL. Cyclosporin A (CsA) was given during all cycles of chemotherapy. We did not observe any severe infections or kidney failure during treatments. The use of conventional cytogenetic analysis plus FISH may identify complex karyotype also in transplanted patients receiving immunotherapy, and may also contribute to a better assessment of PT-AL.

Assignment to chromosome 12q24.33, gene organization and splicing of the human KRAB/FPB containing zinc finger gene ZNF84.

FISH analysis was used to assign the human ZNF84 gene to chromosome 12q24.33, a region associated with recurrent breakpoints and allelic loss in several human cancers. In this report we show that the ZNF84 coding region is organized in four exons; two are dedicated to encoding the KRAB/FPB-A and KRAB/FPB-B modules, the remaining exons encode the N-terminal amino acids and C-terminal array of zinc finger units, respectively.

Molecular cytogenetic resources for chromosome 4 and comparative analysis of phylogenetic chromosome IV in great apes.

We have generated a panel of 55 somatic cell hybrids retaining fragments of human chromosome 4. Each hybrid has been characterized cytogenetically by FISH and molecularly by 37 STSs, evenly spaced along the chromosome. The panel can be exploited to map subregionally DNA sequences on chromosome 4 and to generate partial chromosome paints useful in the characterization of chromosomal rearrangements involving this chromosome. Furthermore, a panel of 84 YACs mapping on chromosome 4 has been characterized by FISH. A subset of this panel is recognized by STSs used in the somatic cell hybrid characterization. In this way a correlation between the genetic and the physical maps can be established. These resources have been used to investigate the conservation of the phylogenetic chromosome IV in great apes. The results indicate that all the pericentric inversions that differentiate chromosome IV in these species are distinct and that one of the breakpoints frequently lies very close to the centromere. In 4 instances, the YAC containing the breakpoint was identified. The breakpoint in IVq of PTR and MMU lies in the same YAC, suggesting that this breakpoint has been utilized twice in the evolutionary history of this chromosome.

Molecular cytogenetic resources specific for chromosome 12.

We have generated a panel of 20 somatic cell hybrids retaining fragments of human chromosome 12. Each hybrid was characterized cytogenetically by reverse fluorescence in situ hybridization (FISH) and molecularly by 24 sequence tagged sites (STSs) spaced evenly along the chromosome. The panel can be exploited to map subregionally DNA sequences on chromosome 12 and to generate partial chromosome paints useful in the characterization of chromosomal rearrangements involving this chromosome. Furthermore, a panel of 58 yeast artificial chromosomes (YACs) mapping to chromosome 12 was characterized by FISH experiments on normal human metaphases. A subset of this panel is recognized by the STSs used in the somatic cell hybrid characterization. In this way a correlation between the genetic and the physical maps of this chromosome can be established.

Evolution of chromosome Y in primates.

We have investigated, by fluorescence in situ hybridization (FISH), the cytogenetic evolution of the Y chromosome in primates using 17 yeast artificial chromosomes, representative of the Y-specific euchromatic region of the human chromosome Y. The FISH experiments were performed on great apes (Homo sapiens, Pan troglodytes, Gorilla gorilla and Pongo pygmaeus pygmaeus), and on two Old World monkeys species as an outgroup (Cercopitecidae Macaca fascicularis and Papio anubis). The results showed that this peculiar chromosome has undergone rapid and unconstrained evolution both in sequence content and organization.

The chemokine receptor CCR8 is preferentially expressed in Th2 but not Th1 cells.

In this paper we report on the cloning and characterization of mouse CCR8. Like its human homologue, it is predominantly expressed in the thymus. In the periphery, murine CCR8 mRNA was found most abundantly expressed in activated Th2-polarized cells and in NK1.1+ CD4+ T cells. Human CCR8 is also preferentially expressed in human Th2-polarized cells and clones. This pattern of expression suggests that CCR8 is part of a Th2-specific gene expression program. The CCR8 ligands I-309 and its mouse homologue T cell activation gene 3 (TCA-3) are potent chemoattractants for Th2-polarized cells. Taken together, these observations strongly suggest that CCR8 plays a role in the control of Th2 responses, and may represent a potential target for treatment of allergic diseases.

Isolation and chromosomal localization of GPR31, a human gene encoding a putative G protein-coupled receptor.

The screening of a human genomic library with a chemokine receptor-like probe allowed us to obtain a putative member of the G protein-coupled receptor gene (GPCR) family, designated GPR31. Its deduced amino acid sequence encodes a polypeptide of 319 amino acids that shares 25-33% homology with members of the chemokine, purino, and somatostatin receptor gene families. Amino acid sequence comparison reveals that the best match in the protein databases is with the human orphan GPCR called HM74 (33% identity). Southern genomic analysis of the GPR31 gene shows a hybridization pattern consistent with that of a single-copy gene. Using fluorescence in situ hybridization, we have determined the chromosomal and regional localization of the GPR31 gene at 6q27. The GPR31 mRNA is expressed at low levels by several human cell lines of different cellular origins. The phylogenetic analysis suggests that the GPR31 receptor may represent a member of a new GPCR subfamily.