ABSTRACT
The t(4;11) translocation is the cytogenetic hallmark of a subset of acute lymphoblastic leukemias characterized by pro-B immunophenotype and a dismal prognosis. This translocation fuses the MLL gene on chromosome band 11q23 and the AF4 gene on 4q21, resulting in the expression of fusion transcripts from both translocated chromosomes. The MLL-AF4 chimeric transcript is thought to mediate the leukemic transformation. The MLL genomic disruption detected by Southern blot and the RT-PCR for the MLL-AF4 chimeric transcript expression are molecular evidence of this chromosomal translocation. However, similar molecular rearrangements have also been identified in cases without the cytogenetic t(4;11). We report a 30-year-old patient with high risk ALL, a normal karyotype, and molecular evidence of MLL-AF4 fusion. Using a double color FISH assay with MLL specific PAC probes, a cryptic t(4;11) due to insertion of 5' MLL sequences in chromosome 4q21 was demonstrated. Consequently the MLL-AF4 was encoded by der(4). This insertion mechanism precludes the genomic recombination of AF4-MLL and supports the crucial role played by MLL-AF4 in leukemogenesis. The findings of our case, along with others, show the importance of complementing the karyotype with molecular and FISH techniques.
Subject(s)
Chromosomes, Human, Pair 11 , Chromosomes, Human, Pair 4 , DNA-Binding Proteins/genetics , Oncogene Proteins, Fusion/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Proto-Oncogenes , Transcription Factors , Translocation, Genetic , Adult , Histone-Lysine N-Methyltransferase , Humans , In Situ Hybridization, Fluorescence , Male , Myeloid-Lymphoid Leukemia ProteinABSTRACT
Reciprocal translocations involving the MLL gene on chromosome band 11q23 have been observed in both acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). In AML, identification of MLL breakpoints is an important prognostic factor. Breakpoints are clustered in an 8 kb DNA fragment (bcr) and can be detected by Southern blotting or fluorescence in situ hybridization (FISH) analysis. Our objective in this study was to design a DNA probe set that enables optimal detection of MLL rearrangements using interphase FISH. Two PAC clones, 217A21 and 167K13, spanning the MLL gene with a minimal overlap in the bcr were isolated and labeled. Twenty-seven AML/ALL patients with cytogenetic 11q23 abnormalities, seven AML/ALL patients without 11q23 abnormalities but MLL rearrangement by Southern blotting, and eight healthy donors were analyzed by FISH. We compared this double-color FISH analysis with FISH using a YAC clone (yB22B2) and with Southern blotting. The PAC probe combination detects an MLL breakpoint in all cases with MLL rearrangement detected by Southern blotting except for cases with a partial tandem duplication detected by reverse transcriptase-polymerase chain reaction (RT-PCR). FISH using the PAC probes also detected MLL breakpoints in four cases with MLL deletions telomeric to the breakpoint that could not be detected by the single probe yB22B2. This new probe set provides a reliable and rapid assay for the diagnosis of AML and ALL patients with MLL/11q23 breakpoints.
Subject(s)
Chromosome Breakage/genetics , Chromosomes, Human, Pair 11/genetics , DNA Probes/genetics , DNA-Binding Proteins/genetics , In Situ Hybridization, Fluorescence/methods , Interphase/genetics , Leukemia/genetics , Proto-Oncogenes , Transcription Factors , Acute Disease , Adolescent , Adult , Aged , Bacteriophage P1/genetics , Child, Preschool , Cloning, Molecular , Contig Mapping , Female , Histone-Lysine N-Methyltransferase , Humans , Karyotyping , Male , Middle Aged , Myeloid-Lymphoid Leukemia Protein , Translocation, Genetic/geneticsABSTRACT
Structural chromosome abnormalities in spermatozoa represent an important category of paternally transmittable genetic damage. A couple was referred to our centre because of repetitive abortions and the man was found to be a carrier of a reciprocal translocation t(3;11)(q27.3;q24.3). A tailored fluorescence in situ hybridisation (FISH) approach was developed to study the meiotic segregation patterns in spermatozoa from this translocation carrier. A combination of three DNA probes was used, a centromeric probe for chromosome 11, a cosmid probe for chromosome 11q and a YAC probe for chromosome 3q. The frequency of spermatozoa carrying an abnormal chromosome constitution was compared with baseline frequencies in control semen specimens and it was found that a significantly higher percentage of spermatozoa carried an abnormal constitution for the chromosomes involved in the translocation. A normal or balanced chromosome constitution was found in 44.3% of the analysed spermatozoa, while the remainder exhibited an abnormal chromosome constitution reflecting different modes of segregation (15.9% adjacent I segregation, 6.5% adjacent II segregation, 28.9% 3:1 segregation, 0.8% 4:0 segregation, 3.6% aberrant segregation). The frequency of aneuploidy for chromosomes X, Y, 13 and 21 was assessed using specific probes but there was no evidence of interchromosomal effects or variations in the sex ratio in spermatozoa from the translocation carrier. In conclusion, structural aberrations can be reliably assessed in interphase spermatozoa using unique DNA probe cocktails, and this method provides insight into the genetic constitution of germ cells and enables evaluation of potential risks for the offspring.
