Common region of ALL-1 gene disrupted in epipodophyllotoxin-related secondary acute myeloid leukemia

Translocations at chromosomal band 11q23 characterize most de novo acute lymphoblastic leukemias (ALL) of infants, acute myeloid leukemias (AML) of infants and young children, and secondary AMLs following epipodophyllotoxin exposure. The chromosomal breakpoints at 11q23 have been cloned from isolated cases of de novo ALL and AML. Using an 859-base pair BamHI fragment of human ALL-1 complementary DNA that recognizes the genomic breakpoint region for de novo ALL and AML, we investigated two cases of secondary AML that followed etoposide-treated primary B-lineage ALL. In the first case, the translocation occurred between chromosomes 9 and 11 and the breakpoint at 11q23 localized to the same 9-kilobase region of the ALL-1 gene that is disrupted in most of the de novo leukemias. In the second case the translocation was between chromosomes 11 and 19. The breakpoint occurred outside of the ALL-1 breakpoint cluster region.     

Secondary leukemias induced by topoisomerase-targeted drugs

The major established cause of acute myeloid leukemia (AML) in the young is cancer chemotherapy. There are two forms of treatment-related AML (t-AML). Each form has a de novo counterpart. Alkylating agents cause t-AML characterized by antecedent myelodysplasia, a mean latency period of 5-7 years and complete or partial deletion of chromosome 5 or 7. The risk is related to cumulative alkylating agent dose. Germline NF-1 and p53 gene mutations and the GSTT1 null genotype may increase the risk. Epipodophyllotoxins and other DNA topoisomerase II inhibitors cause leukemias with translocations of the MLL gene at chromosome band 11q23 or, less often, t(8;21), t(3;21), inv(16), t(8;16), t(15;17) or t(9;22). The mean latency period is about 2 years. While most cases are of French-American-British (FAB) M4 or FAB M5 morphology, other FAB AML subtypes, myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL) and chronic myelogenous leukemia (CML) occur. Between 2 and 12% of patients who receive epipodophyllotoxin have developed t-AML. There is no relationship with higher cumulative epipodophyllotoxin dose and genetic predisposition has not been identified, but weekly or twice-weekly schedules and preceding l-asparaginase administration may potentiate the risk. The translocation breakpoints in MLL are heterogeneously distributed within a breakpoint cluster region (bcr) and the MLL gene translocations involve one of many partner genes. DNA topoisomerase II cleavage assays demonstrate a correspondence between DNA topoisomerase II cleavage sites and the translocation breakpoints. DNA topoisomerase II catalyzes transient double-stranded DNA cleavage and rejoining. Epipodophyllotoxins form a complex with the DNA and DNA topoisomerase II, decrease DNA rejoining and cause chromosomal breakage. Furthermore, epipodophyllotoxin metabolism generates reactive oxygen species and hydroxyl radicals that could create abasic sites, potent position-specific enhancers of DNA topoisomerase II cleavage. One proposed mechanism for the translocations entails chromosomal breakage by DNA topoisomerase II and recombination of DNA free ends from different chromosomes through DNA repair. With few exceptions, treatment-related leukemias respond less well to either chemotherapy or bone marrow transplantation than their de novo counterparts, necessitating more innovative treatments, a better mechanistic understanding of the pathogenesis, and strategies for prevention.     

Risk factors for serious nonsteroidal-induced gastrointestinal complications: regression analysis of the MUCOSA trial

Gene rearrangements involving MLL (also known as ALL1, HRX, or Htrx) are among the most common molecular abnormalities associated with acute leukemia. These leukemias generally have one allele involved in a rearrangement, while the remaining allele is uninvolved and demonstrates a germline MLL configuration. In this study, we describe a leukemic cell line that does not have a germline MLL allele and thus cannot produce a normal MLL gene product. We show that the ML-1 cell line, derived from a patient with acute myeloid leukemia, has one allele involved in a t(6;11)(q27;q23), while the remaining MLL allele is deleted. Cloning of the genomic breakpoints on the derivative(6) and der(11) chromosomes demonstrated a balanced translocation between MLL on chromosome band 11q23 and AF6 on chromosome band 6q27. Sequence analysis of the derivative chromosomes revealed that a 186-bp segment of MLL intron 6, downstream of the breakpoint, had been duplicated, inverted, and inserted between MLL and AF6 on the der(11) chromosome. In light of the fact that ML-1 cells can be induced to differentiate along the granulocyte and macrophage lineages, the finding that ML-1 lacks a germline MLL allele demonstrates that a normal MLL gene is not required for survival, proliferation, or differentiation of this cell line.     

Fusion of the MLL gene with two different genes, AF-6 and AF-5alpha, by a complex translocation involving chromosomes 5, 6, 8 and 11 in infant leukemia

We analysed a complex translocation involving chromosomes 5, 6, 8 and 11 in a case of infant leukemia. Molecular analysis of the MLL gene revealed that MLL was fused with two different genes, AF-6 on chromosome 6q27 and AF-5alpha. AF-5alpha, the 11th partner gene fused with MLL, is a novel gene mapped to chromosome 5q12, which encodes a 31 kDa protein of 269 amino acids and contains a possible nuclear targeting sequence, a potential leucine zipper dimerization motif and an alpha-helical coiled-coil domain. In situ hybridization and molecular cloning analyses demonstrated that two different types of chromosomal recombination had occurred in the cells. One was a three-way translocation among chromosomes 6, 8 and 11, and the other was an insertion of a chromosome 5-derived segment into the breakpoint of chromosomes 8 and 11. Accordingly, the karyotype was defined as del(5)(q11.2q12), der(6)t(6;8) (q27;q11.2), der(8)(8pter-->8q11.2::5q11.2-->5q12::11q23-->++ +11qter), der(11)t(6;11) (q27;q23). Thus, the MLL gene created two different fusion mRNAs, since the chromosome 11 split into two different chromosomes 5 and 6. This is the first report demonstrating fusion of the MLL gene with two different genes by a complex translocation.     

Chimeric MLL products with a Ras binding cytoplasmic protein AF6 involved in t(6;11) (q27;q23) leukemia localize in the nucleus

In infantile leukemias and therapy-related leukemias, the MLL gene is frequently found to be disrupted and fused to various translocation partner genes, such as AF4/FEL, LTG9/AF9 and LTG19/ENL as a result of 11q23 translocations. We previously showed that the N-terminal portion common to various chimeric MLL products, as well as to MLL-LTG9 and MLL-LTG19, localizes in the nuclei, and therefore suggested that it might play an important role in leukemogenesis. In the present study, MLL-AF6 chimeric products found in the t(6;11)(q27;q23) translocation were analysed since AF6, a Ras-binding protein, exhibits a different subcellular localization from that of LTG9/AF9 and LTG19/ENL. Immunofluorescence staining data and cell fractionation analyses demonstrated that MLL-AF6 chimeric products localize in the nuclei despite the fact that AF6 itself localizes in the cytoplasm, confirming the importance of the nuclear localization of chimeric MLL products. The region in the N-terminal portion of MLL responsible for this nuclear localization was examined and found to be a region containing AT-hook motifs.     

The behavior of late potentials in myocardial infarct patients undergoing myocardial revascularization

A novel 30 kb deletion of the beta-globin gene cluster associated with the phenotype of hereditary persistence of fetal hemoglobin (HPFH) is described in two unrelated individuals of Vietnamese background. The Vietnamese G gamma A gamma HPFH deletion has a unique 5' breakpoint 3.5 kb downstream of the delta-globin gene. The 3' breakpoint lies approximately 8 kb upstream from the HPFH-3 breakpoint (Henthorn et al., 1986) and in the region of the 3' breakpoints of HPFH-4 (Saglio et al., 1986), German and Belgian G gamma+ (A gamma delta beta)zero-thalassemias (Anagnou et al., 1988; Losekoot et al., 1991). Characterisation of the 3' breakpoint in the present study has enabled more precise localisation of other deletion breakpoints at this locus. Further evidence is provided that the 3' breakpoint region contains functionally important sequences and that the juxtaposition of these sequences to the gamma-globin genes is a significant factor in the increased fetal hemoglobin levels.     

Potential role for wild-type p53 in leukemias with MLL gene translocations

We used single-strand conformation polymorphism (SSCP) analysis of p53 exons 4-8 to screen for possible mutations in 25 pediatric de novo leukemias with translocations of the MLL gene at chromosome band 11q23. Of the 25 patients, 21 were infants. Fifteen cases were acute myeloid leukemia (AML), eight were acute lymphoblastic leukemia (ALL), and two cases were biphenotypic. Nineteen cases were studied at diagnosis and six at time of relapse. p53 mutations were absent in all 19 cases studied at the time of diagnosis. The only mutation was a TGC-->TTC transversion (cys-->phe) at codon 141 in exon 5 in a case of infant ALL at relapse that occurred by subclone evolution after MLL gene translocation. We previously showed that p53 mutations are also absent in pediatric treatment-related leukemias with MLL gene translocations. The absence of p53 mutations at initial transformation may suggest that the anti-apoptotic effect of mutant p53 is not important in leukemias with MLL gene translocations. Alternatively, exogenous DNA damage may be the common feature in treatment-related and de novo cases. Since MLL gene translocations may occur through DNA repair and wild-type p53 is central to DNA repair, the absence of p53 mutations raises the possibility that wild-type p53, not mutant p53, may be important in the genesis of leukemias with these translocations.     

The structure of the human ALL-1/MLL/HRX gene

The human ALL-1/MLL/HRX gene on chromosome 11q23 is the site of many locally clustered chromosomal alterations associated with several types of acute leukemias, including deletions. partial duplications and reciprocal translocations. Structurally variant proteins derived from an altered ALL-1 gene presumably make essential contributions to the malignant transformation of hematopoietic progenitor cells. The ALL-1 gene is spread over approximately 92 kb and consists of at least 37 exons. An exon/intron map including the position of the 3'-end of the gene and a detailed restriction map were produced and an updated map is presented. Data from other laboratories were incorporated where compatible. Exon/intron boundaries were sequenced and an intron-phase analysis was performed. The results are expected to contribute to a better understanding of those structural alterations of the gene that conserve the open reading frame and produce presumably oncogenic variants of the ALL-1 protein. They will also facilitate the rapid molecular diagnosis of structural alterations of this gene and the choice of therapeutic options. Mechanisms that may potentially account for the striking clustering of the translocation breakpoints in the breakpoint cluster region of the gene are discussed.     

Analysis of 1;17 translocation breakpoints in neuroblastoma: implications for mapping of neuroblastoma genes

Deletions and translocations resulting in loss of distal 1p-material are known to occur frequently in advanced neuroblastomas. Fluorescence in situ hybridisation (FISH) showed that 17q was most frequently involved in chromosome 1p translocations. A review of the literature shows that 10 of 27 cell lines carry 1;17 translocations. Similar translocations were also observed in primary tumours. Together with the occurrence of a constitutional 1;17 translocation in a neuroblastoma patient, these observations suggest a particular role for these chromosome re-arrangements in the development of neuroblastoma. Apart from the loss of distal 1p-material, these translocations invariably lead to extra copies of 17q. This also suggested a possible role for genes on 17q in neuroblastoma tumorigenesis. Further support for this hypothesis comes from the observation that in those cell lines without 1;17 translocations, other chromosome 17q translocations were present. These too lead to extra chromosome 17q material. Molecular analysis of 1;17 translocation breakpoints revealed breakpoint heterogeneity both on 1p and 17q, which suggests the involvement of more than 2 single genes on 1p and 17q. The localisation of the different 1p-breakpoints occurring in 1;17 translocations in neuroblastoma are discussed with respect to the recently identified candidate tumor suppressor regions and genes on 1p. In this study, we focused on the molecular analysis of the 17q breakpoints in 1;17 translocations. Detailed physical mapping of the constitutional 17q breakpoint allowed for the construction of a YAC contig covering the breakpoint. Furthermore, a refined position was determined for a number of 17q breakpoints of 1;17 translocations found in neuroblastoma cell lines. The most distal 17q breakpoint was identified in cell line UHG-NP and mapped telomeric to cosmid cCI17-1049 (17q21). This suggests that genes involved in a dosage-dependent manner in the development of neuroblastoma map in the distal segment 17q22-qter. Future studies aim at the molecular cloning of 1;17 translocation breakpoints and at deciphering the mechanisms leading to 1;17 translocations and possibly to the identification of neuroblastoma genes at or in the vicinity of these breakpoints.     

Adaptation to the mental health setting: the lived experience of comprehensive nurse graduates

Karyotype analysis of a primary culture from a case of papillary thyroid cancer (PTC) showed an abnormal short arm of one homologue of chromosome 2 as sole abnormality in 4 of 16 metaphases. Based on G-banding analysis, two different aberration types on chromosome 2 could be assumed representing either a del(2)(p22-23) or a pericentric inversion. Further comparative genomic hybridization (CGH) analysis as well as fluorescence in situ hybridization (FISH) analysis were performed to confirm the assumed alterations. While CGH analysis showed no loss of chromosome 2 material, FISH with yeast artificial chromosome (YAC) probes homologous to the region 2p22-23 demonstrated two pericentric inversions of chromosome 2 involving different breakpoints on 2p in 6.8% and 4.2% of the metaphases, respectively. Polymerase chain reaction (PCR) analysis with degenerated oligonucleotide primers that bind within the conserved catalytic domain of tyrosine kinase (tk) genes resulted in amplification products with DNA of YAC 851D11 suggesting the presence of such genes at or near the translocation breakpoint.