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Cloning the breakpoint cluster region of the inv(16) in acute nonlymphocytic leukemia M4 Eo
J G Dauwerse1, J W Wessels, R H Giles
1Department of Human Genetics, Leiden University, Sylvius Laboratories, The Netherlands.
Abstract:
The pericentric inversion of chromosome 16 and the t(16;16) are two recurrent aberrations in bone marrow of patients with acute nonlymphocytic leukemia subtype M4 Eo, characterized by abnormal eosinophilic granulation. We describe here the precise localization of the breakpoints using fluorescence in situ hybridization (FISH) with cosmids spread over the short arm of chromosome 16 and the detection, isolation and characterization of a 14Kb EcoRI fragment containing a cluster of breakpoints. First, cosmids were mapped to intervals defined by constitutional 16p rearrangements, second, the inv(16) and t(16;16) breakpoints were mapped to one of the intervals using FISH with the mapped cosmids and third, cosmids within this interval were ordered using two color interphase FISH. An STS of the cosmid closest to the breakpoints was then used to isolate five YACs, which did span all of the 16 inv(16) breakpoints and one t(16;16) breakpoint analysed. In the DNA of one inv(16) patient we detected an additional submicroscopic deletion immediately proximal to the 16p breakpoint. Since this patient has the same phenotype, the 16p sequences proximal to the breakpoint seem non-essential to M4 Eo. This implies that the pathologic event is the juxtaposition of sequences distal to the 16p breakpoint with sequences proximal to the 16q breakpoint. While four of the five YACs showed instability of the region around the inv(16) breakpoint, DNA halo analysis allowed us to identify one YAC which was co-linear with normal genomic DNA and has yielded the actual breakpoint sequences which could be subcloned into cosmids and fosmids.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Recurrent chromosome 16 aberrations in acute myeloid leukemia M4 Eo involve breakpoints on chromosome 16p. These rearrangements are crucial for the disease, suggesting a pathogenic juxtaposition of genetic sequences.
Area of Science:
- Cytogenetics
- Molecular Biology
- Hematologic Oncology
Background:
- Recurrent chromosomal aberrations, specifically pericentric inversion of chromosome 16 (inv(16)) and t(16;16), are hallmarks of acute myeloid leukemia subtype M4 Eo.
- This subtype is characterized by abnormal eosinophilic granulation in bone marrow cells.
Purpose of the Study:
- To precisely localize the breakpoints associated with inv(16) and t(16;16) on chromosome 16p.
- To identify and characterize the specific DNA sequences involved in these recurrent chromosomal rearrangements.
Main Methods:
- Fluorescence in situ hybridization (FISH) using chromosome 16p cosmids to map breakpoints.
- Ordering cosmids via two-color interphase FISH.
- Isolation and characterization of breakpoint cluster regions using Yeast Artificial Chromosomes (YACs).
- DNA halo analysis to identify stable YAC clones for breakpoint sequencing.
Main Results:
- Precise localization of breakpoints for inv(16) and t(16;16) on chromosome 16p.
- Identification of a 14Kb EcoRI fragment containing a cluster of breakpoints.
- Detection of a submicroscopic deletion proximal to the 16p breakpoint in one patient, suggesting non-essentiality of these proximal sequences for M4 Eo.
- Isolation of a stable YAC clone yielding breakpoint sequences, enabling subcloning.
Conclusions:
- The pathogenic event in M4 Eo associated with inv(16) and t(16;16) likely involves the juxtaposition of genetic sequences distal to the 16p breakpoint with those proximal to the 16q breakpoint.
- Sequences proximal to the 16p breakpoint appear non-essential for the M4 Eo phenotype.
- The identified breakpoint sequences provide a basis for further molecular characterization of these leukemia subtypes.