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Biomedical subjects

C A Felix

Publications and source records attributed to C A Felix.

At least 37 records · Page 2Linked to original sources
PubMed1998

Association of germline p53 mutation with MLL segmental jumping translocation in treatment-related leukemia.

Segmental jumping translocations are chromosomal abnormalities in treatment-related leukemias characterized by multiple copies of the ABL and/or MLL oncogenes dispersed throughout the genome and extrachromosomally. Because gene amplification potential accompanies loss of wild-type p53, we examined the p53 gene in a case of treatment-related acute myeloid leukemia (t-AML) with MLL segmental jumping translocation. The child was diagnosed with ganglioneuroma and embryonal rhabdomyosarcoma (ERMS) at 2 years of age. Therapy for ERMS included alkylating agents, DNA topoisomerase I and DNA topoisomerase II inhibitors, and local radiation. t-AML was diagnosed at 4 years of age. The complex karyotype of the t-AML showed structural and numerical abnormalities. Fluorescence in situ hybridization analysis showed multiple copies of the MLL gene, consistent with segmental jumping translocation. A genomic region including CD3, MLL, and a segment of band 11q24 was unrearranged and amplified by Southern blot analysis. There was no family history of a cancer predisposing syndrome, but single-strand conformation polymorphism (SSCP) analysis detected identical band shifts in the leukemia, ganglioneuroma, ERMS, and normal tissues, consistent with a germline p53 mutation, and there was loss of heterozygosity in the ERMS and the t-AML. Sequencing showed a CGA-->TGA nonsense mutation at codon 306 in exon 8. The results of this analysis indicate that loss of wild-type p53 may be associated with genomic instability after DNA-damaging chemotherapy and radiation, manifest as a complex karyotype and gene amplification in some cases of t-AML.

Acute Disease↗
PubMed1998

t(11;22)(q23;q11.2) In acute myeloid leukemia of infant twins fuses MLL with hCDCrel, a cell division cycle gene in the genomic region of deletion in DiGeorge and velocardiofacial syndromes.

We examined the MLL genomic translocation breakpoint in acute myeloid leukemia of infant twins. Southern blot analysis in both cases showed two identical MLL gene rearrangements indicating chromosomal translocation. The rearrangements were detectable in the second twin before signs of clinical disease and the intensity relative to the normal fragment indicated that the translocation was not constitutional. Fluorescence in situ hybridization with an MLL-specific probe and karyotype analyses suggested t(11;22)(q23;q11. 2) disrupting MLL. Known 5' sequence from MLL but unknown 3' sequence from chromosome band 22q11.2 formed the breakpoint junction on the der(11) chromosome. We used panhandle variant PCR to clone the translocation breakpoint. By ligating a single-stranded oligonucleotide that was homologous to known 5' MLL genomic sequence to the 5' ends of BamHI-digested DNA through a bridging oligonucleotide, we formed the stem-loop template for panhandle variant PCR which yielded products of 3.9 kb. The MLL genomic breakpoint was in intron 7. The sequence of the partner DNA from band 22q11.2 was identical to the hCDCrel (human cell division cycle related) gene that maps to the region commonly deleted in DiGeorge and velocardiofacial syndromes. Both MLL and hCDCrel contained homologous CT, TTTGTG, and GAA sequences within a few base pairs of their respective breakpoints, which may have been important in uniting these two genes by translocation. Reverse transcriptase-PCR amplified an in-frame fusion of MLL exon 7 to hCDCrel exon 3, indicating that an MLL-hCDCrel chimeric mRNA had been transcribed. Panhandle variant PCR is a powerful strategy for cloning translocation breakpoints where the partner gene is undetermined. This application of the method identified a region of chromosome band 22q11.2 involved in both leukemia and a constitutional disorder.

Abnormalities, Multiple↗
PubMed1998

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.

Child↗
PubMed1998

Panhandle PCR: a technical advance to amplify MLL genomic translocation breakpoints.

Translocations involving a breakpoint cluster region of the MLL gene at chromosome band 11q23 are the most common molecular abnormalities in acute leukemias of infants and acute leukemias related to chemotherapy with DNA topoisomerase II inhibitors. Molecular cloning of MLL genomic breakpoints by PCR has previously been difficult because MLL has many translocation partners and several breakpoints involve unknown partner genes. We review a new approach to MLL genomic breakpoint cloning called panhandle PCR. By adding an oligonucleotide sequence to the unknown 3' partner gene that is complementary to a known 5' MLL sequence, we have been able to generate a genomic template with an intrastrand loop for PCR schematically shaped like a pan with a handle. The intrastrand loop contains the translocation breakpoint and unknown partner DNA, while the handle contains the known 5' sequence from MLL and a complement to that sequence. Primers both derived from MLL are used to amplify the breakpoint by panhandle PCR. Panhandle PCR offers the advantage of having specificity for the strand of interest at both primer annealing sites without requiring specific primers for the many partner genes of MLL. Panhandle PCR is a straightforward method that represents a technical advance in MLL genomic breakpoint cloning.

Chromosome Deletion↗
PubMed1998

Overexpression of p53 protein in cutaneous T cell lymphoma: relationship to large cell transformation and disease progression.

The molecular mechanisms by which advanced cases of cutaneous T cell lymphoma (CTCL) (mycosis fungoides/Sezary syndrome) undergo large cell transformation (LCT) and develop the morphologic appearance of a large cell lymphoma, are undefined. We used immunohistochemical analysis and polymerase chain reaction/single strand conformational polymorphism to examine whether p53 mutations are associated with disease progression and LCT in CTCL. p53 protein immunohistochemistry was performed on 37 paraffin embedded biopsies from 27 patients with CTCL; LCT was present in 15 biopsies. Overexpression of p53 protein was found in 11 of 37 CTCL biopsies including 10 of 15 biopsies (67%) with LCT in which p53 staining was predominantly seen in large transformed cells. In contrast, p53 immunostaining was found in only one of 22 CTCL biopsies without LCT (p < 0.0004). Serial biopsies revealed acquisition of p53 expression following LCT in two patients in whom initial diagnostic biopsies without LCT were p53 negative by immunostaining. All p53 protein positive biopsies were from advanced lesions (cutaneous tumors or extracutaneous sites); none of 12 patch/plaque stage CTCL biopsies demonstrated p53 staining. Polymerase chain reaction/single strand conformational polymorphism and sequencing analysis of p53 exons 4-8 was performed in 11 cases where frozen tissue was available. No mutations were detected in six cases positive for p53 protein expression. These results suggest overexpression of p53 protein in LCT and disease progression of CTCL by a mechanism other than p53 gene mutation, in most cases.

Base Sequence↗
PubMed1998

RAS mutations in pediatric leukemias with MLL gene rearrangements.

Translocations of the MLL gene at chromosome band 11q23 are the most common cytogenetic alterations in de novo leukemia in infants and in leukemia related to chemotherapy with DNA topoisomerase II inhibitors. Experiments on knock-in mice suggest that additional mutational events may by required for full leukemogenesis. Therefore, we used single-strand conformation polymorphism analysis and an allele-specific restriction enzyme assay to investigate the frequency of KRAS and NRAS mutations in 32 pediatric leukemias with translocation of the MLL gene. Of 25 de novo cases, 13 were acute lymphoblastic leukemia (ALL), 10 were acute myeloid leukemia (AML), and 2 were biphenotypic. Three secondary leukemias were AML, 1 was biphenotypic, 1 was ALL, and 2 were diagnosed as myelodysplasia. The frequency of RAS mutations was 2 of 10 in de novo AML. Both mutations occurred in infant monoblastic variants. RAS mutations were otherwise absent in this series. This is the first report of congenital leukemias where translocation of the MLL gene and RAS mutation coexist. The frequency of RAS mutations in de novo AMLs with MLL gene translocations is similar to that in other forms of AML, but RAS mutations play a limited role in lymphoid and treatment-related leukemias with similar translocations.

Adolescent↗
PubMed1997

Panhandle polymerase chain reaction amplifies MLL genomic translocation breakpoint involving unknown partner gene.

We used a new approach called panhandle polymerase chain reaction (PCR) to clone an MLL genomic translocation breakpoint in a case of acute lymphoblastic leukemia of infancy in which karyotype analysis was technically unsuccessful and did not show the translocation partner. Panhandle PCR amplified known MLL sequence 5' of the breakpoint and 3' sequence from the unknown partner gene from a DNA template with an intrastrand loop schematically shaped like a pan with a handle. The 7-kb panhandle PCR product contained the translocation breakpoint in MLL intron 8. The partner DNA included unique nonrepetitive sequences, Alu and mammalian apparent LTR-retrotransposon (MaLR) repetitive sequences, and a region of homology to expressed sequence tags. MaLR sequences have not been found before near leukemia-associated translocation breakpoints. The nonrepetitive sequences were not homologous to known partner genes of MLL. Screening of somatic cell hybrid and radiation hybrid lines by PCR and fluorescence in situ hybridization analysis of normal metaphase chromosomes mapped the partner DNA to chromosome band 4q21. Reverse transcriptase-PCR identified an MLL-AF-4 chimeric mRNA, indicating that panhandle PCR identified a fusion of MLL with a previously uncharacterized AF-4 intronic sequence. Panhandle PCR facilitates cloning translocation breakpoints and identifying unknown partner genes.

Base Sequence↗
PubMed1997

Panhandle PCR strategy to amplify MLL genomic breakpoints in treatment-related leukemias.

Panhandle PCR amplifies genomic DNA with known 5' and unknown 3' sequences from a template with an intrastrand loop schematically shaped like a pan with a handle. We used panhandle PCR to clone MLL genomic breakpoints in two pediatric treatment-related leukemias. The karyotype in a case of treatment-related acute lymphoblastic leukemia showed the t(4;11)(q21;q23). Panhandle PCR amplified the translocation breakpoint at position 2158 in intron 6 in the 5' MLL breakpoint cluster region (bcr). The karyotype in a case of treatment-related acute myeloid leukemia was normal, but Southern blot analysis showed a single MLL gene rearrangement. Panhandle PCR amplified the breakpoint at position 1493 in MLL intron 6. Screening of somatic cell hybrid and radiation hybrid DNAs by PCR and reverse transcriptase-PCR analysis of the leukemic cells indicated that panhandle PCR identified a fusion of MLL intron 6 with a previously uncharacterized sequence in MLL intron 1, consistent with a partial duplication. In both cases, the breakpoints in the MLL bcr were in Alu repeats, and there were Alu repeats in proximity to the breakpoints in the partner DNAs, suggesting that Alu sequences were relevant to these rearrangements. This study shows that panhandle PCR is an effective method for cloning MLL genomic breakpoints in treatment-related leukemias. Analysis of additional pediatric cases will determine whether breakpoint distribution deviates from the predilection for 3' distribution in the bcr that has been found in adult cases.

Adult↗
PubMed1996

The p53 gene in pediatric therapy-related leukemia and myelodysplasia.

We investigated the frequency of p53 mutations in 19 pediatric cases of therapy-related leukemia or myelodysplastic syndrome. Eleven children presented with acute myeloid leukemia, one with mixed-lineage leukemia, two with acute lymphoblastic leukemia, and five with myelodysplasia at times ranging from 11 months to 9 years after a primary cancer diagnosis. The primary cancers, which included 11 solid tumors and eight leukemias, were treated with various combinations of DNA topoisomerase II inhibitors, alkylating agents, or irradiation. Leukemic or myelodysplastic marrows were screened for possible mutations by single-strand conformation polymorphism (SSCP) analysis of p53 exons 4 to 8. The only observed mutation was an inherited 2-basepair deletion at codon 209 in exon 6 that would shift the open reading frame, create a premature termination codon, and foreshorten the resultant protein. Prior therapy in this patient included DNA topoisomerase II inhibitors, alkylating agents, and irradiation. The secondary leukemia presented as myelodysplasia with monosomies of chromosomes 5 and 7 and abnormalities of chromosome 17. Although the primary cancer was an embryonal rhabdomyosarcoma and there was a family history of cancer, the case did not fulfill the clinical criteria for Li-Fraumeni syndrome. This study suggests that germline p53 mutations may predispose some children to therapy-related leukemia and myelodysplasia, but that p53 mutations otherwise are infrequent in this setting.

Adolescent↗
PubMed1995

Clonal expansion of germline B-lineage acute lymphoblastic leukemia in severe combined immunodeficient mice.

CD19+ B lineage acute lymphoblastic leukemias (ALLs) with unrearranged Ig and TCR genes are designated germline B lineage ALLs. We used CDR3 PCR to determine whether pediatric germline B lineage ALLs contain minor subclones with rearranged Ig H V genes. In six of seven cases there were no PCR detectable CDR3 rearrangements. One case with a smear pattern on CDR3 PCR contained multiple unique CDR3 sequences at frequencies of 1-2 per 2,600, suggesting that polyclonal B cells were present at low frequency. To verify that the germline patterns were from leukemic cells and evaluate in vivo subclone differentiation, a germline B lineage ALL with the t(4;11) translocation was propagated in severe combined immunodeficient SCID) mice. The Ig and TCR genes in the leukemic cells recovered from mouse tissues were germline by Southern blot analysis except for single rearrangements that suggested subclone evolution at the Ig H and lambda loci in addition to the germline population. No CDR3 sequences were detected, indicating that the observed Ig H gene rearrangement most likely was a DJ joining. This study suggests that the transformed cell in germline B lineage ALL represents an early pro-B cell with limited tendency to further differentiate.

Adolescent↗
PubMed1995

Chromosome band 11q23 translocation breakpoints are DNA topoisomerase II cleavage sites.

Human leukemias with 11q23 translocations occur sporadically and after cancer treatment with DNA topoisomerase II-targeted drugs. To investigate this process, we examined DNA topoisomerase II cleavage in vitro in subclones of the normal 11q23 genomic homologue and a t(9;11) translocation breakpoint junction. Cleavage was assayed with limiting dilutions of enzyme in the presence or absence of epipodophyllotoxin and ATP. The strongest sites of cleavage coincided with the t(9;11) breakpoint site and two other translocation breakpoint sites within the normal homologue. These results support the involvement of DNA topoisomerase II in the translocation process at chromosome band 11q23.

Base Sequence↗
PubMed1995

Complex replication error causes p53 mutation in a Li-Fraumeni family.

We demonstrated a germline p53 replication error in two generations of a Li-Fraumeni family affected with liposarcoma, adrenocortical carcinoma, and osteosarcoma. The trinucleotide repeat mutation changed 5'-AGT GTG GTG GTG-3' at codons 215-218 to 5'-AGT TGG TTG GTG GTG-3'. The predicted protein would be elongated by one amino acid (val216-->trp leu) without a change in charge. Detection of p53 in the adrenal tumor by immunostaining suggested that the mutant protein was expressed. Persistence of the mutation in the germline may suggest a defect in DNA repair in the family member first affected. This is the first report where germline transmission of replication-damaged p53 trinucleotide repeats is associated with the Li-Fraumeni syndrome.

Adrenal Cortex Neoplasms↗
PubMed1995

ALL-1 gene rearrangements in DNA topoisomerase II inhibitor-related leukemia in children.

We examined clinical, morphologic, and cytogenetic features and ALL-1 (MLL, Htrxl, HRX) gene rearrangements in 17 cases of secondary leukemia that occurred 11 months to 9 years from diagnoses of primary cancers in children who received topoisomerase II inhibitors or developed secondary leukemias typical of those associated with this therapy. Primary diagnoses included nine solid tumors and eight leukemias. Ten secondary leukemias were acute myeloid leukemia (AML), one was of mixed lineage, two were acute lymphoblastic leukemia (ALL), and four presented as myelodysplasia. Of 15 cases with 11q23 involvement, 11 (73%) were cytogenetically identifiable; four cases had molecular rearrangement only. By Southern blot, rearrangements within the ALL-1 gene were similar to sporadic cases. The results of this analysis suggest the following: (1) In most pediatric cases of topoisomerase II inhibitor-associated leukemia, there is disruption of the breakpoint cluster region of the ALL-1 gene at chromosomal band 11q23. (2) Exposure histories vary in secondary 11q23 leukemia, as the only topoisomerase II inhibitor was dactinomycin in one case, and, in another case, no topoisomerase II inhibitor was administered. (3) There is clinical, morphologic, cytogenetic, and molecular heterogeneity in pediatric secondary 11q23 leukemia. (4) There are some survivors of pediatric secondary 11q23 leukemia, but the outcome is most often fatal.

Acute Disease↗
PubMed1995

p53 gene mutations in pediatric brain tumors.

We investigated the frequency of p53 mutations in 47 pediatric brain tumors of various histologic subtypes that were collected over a period of 5 years. The specimens included 15 primitive neuroectodermal tumors (PNETs), 17 low grade astrocytomas, one anaplastic astrocytoma, three glioblastomas (GBMs), one mixed glial tumor, eight ependymomas, one choroid plexus carcinoma, and one gangliocytoma/ganglioneuroma. Mutations were identified by single strand conformation polymorphism analysis of exons 4-8 and verified by sequencing. Mutations were present in 2 of 3 cases of GBM, but not in 17 low grade astrocytomas (P = 0.02, Fisher's exact test). One GBM demonstrated a germline GGC to AGC transition (gly to ser) at codon 245 with loss of the wild-type allele. A second GBM contained a CGG to TGG transition (arg to trp) at codon 248, also with loss of the wild-type allele, but normal tissue was not available for comparison. In addition, one of 15 PNETs retained heterozygosity but demonstrated a somatic CGT to TGT transition (arg to cys) at codon 273. p53 mutations were absent in other histologic subtypes and in two cases with multiple primary cancers. These data are consistent with earlier findings that p53 mutations are rare in PNETs, which are primarily pediatric tumors. In contrast to adult gliomas, p53 mutations in pediatric gliomas appear restricted to the GBMs. The lack of p53 mutations in pediatric low grade astrocytomas suggests not only histological differences, but also a different molecular pathogenesis in adult and pediatric patients.

Adolescent↗
PubMed1994

Differentiation stages of childhood acute lymphoblastic leukemias with p53 mutations.

Based upon in vitro evidence of p53 involvement in lymphoid differentiation, we assessed immunoglobulin (Ig) and T-cell receptor (TCR) genes in five acute lymphoblastic leukemias (ALLs) with, and 24 ALLs without p53 mutations to compare their genotypic stages. Using Southern blot analysis and complementarity determining region III polymerase chain reaction (CDRIII PCR), 18 cases of B-lineage ALL and 11 cases of T-ALL were studied. Of 20 specimens from 18 B-lineage ALLs, two of four with p53 mutation and two of 16 without mutation had an unrearranged Ig and TCR genotype (p = 0.16; Fisher's exact test). Of 11 cases of T-ALL, the one case with p53 mutation had a rearranged TCR and Ig genotype and a case without mutation was unrearranged. The study indicates that p53 mutation is an infrequent feature of ALL found, nonetheless, in every genotypic subset. The p53 mutations in cases that do not further rearrange may support p53 involvement in lymphoid differentiation, but the heterogeneity in differentiation stages in cases both with and without p53 mutations suggests that regulation of early lymphoid maturation is multifactorial.

Adolescent↗
PubMed1994

Polymorphism at codon 36 of the p53 gene.

A polymorphism at codon 36 in exon 4 of the p53 gene was identified by single strand conformation polymorphism (SSCP) analysis and direct sequencing of genomic DNA PCR products. The polymorphic allele, present in the heterozygous state in genomic DNAs of four of 100 individuals (4%), changes the codon 36 CCG to CCA, eliminates a FinI restriction site and creates a BccI site. Including this polymorphism there are four known polymorphisms in the p53 coding sequence.

Base Sequence↗
PubMed1993

Potential topoisomerase II DNA-binding sites at the breakpoints of a t(9;11) chromosome translocation in acute myeloid leukemia.

We have examined a t(9;11)(p22;q23) chromosome translocation in an acute myeloid leukemia of an infant. The breakpoints on the two chromosomes occurred within introns of the involved genes: AF-9 on chromosome 9, and ALL-1 on chromosome 11. Sequence analysis identified heptamers flanking the breakpoints on both chromosomes 9 and 11, suggesting that the V-D-J recombinase was involved in the translocation. The presence of an N-region between the two chromosomes supports the hypothesis that a mistake in V-D-J joining was involved in the genesis of the translocation and indicates that terminal deoxynucleotidyl transferase was expressed in the cells from which this acute myeloid leukemia originated. In addition, potential topoisomerase II DNA-binding sites were found near the breakpoints of both chromosomes, suggesting the involvement of altered topoisomerase II activity in this translocation. Altered topoisomerase II activity in the presence of an active V-D-J recombinase may be a pathogenetic mechanism of acute myeloid leukemia with rearrangements at 11q23.

Amino Acid Sequence↗