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ETV6-AML1 translocation breakpoints cluster near a purine/pyrimidine repeat region in the ETV6 gene
S P Thandla1, J E Ploski, S Z Raza-Egilmez
1Departments of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
Insights
The ETV6-AML1 fusion gene, common in pediatric leukemia, involves breakpoints in ETV6 intron 5 and AML1 intron 1. AML1 breakpoints are dispersed, unlike clustered ETV6 breakpoints, suggesting non-V(D)J recombination mechanisms.
Area of Science:
- Genetics
- Molecular Biology
- Hematology
Background:
- The t(12;21)(p13;q22) translocation creates the ETV6-AML1 fusion gene, a hallmark of pediatric B-cell precursor acute lymphoblastic leukemia (B-ALL).
- Previous studies characterized the ETV6 breakpoint cluster region (bcr) in intron 5, but the large size and unknown structure of AML1 intron 1 hindered breakpoint mapping.
Purpose of the Study:
- To map the AML1 gene and its breakpoints within intron 1.
- To analyze the ETV6-AML1 breakpoint junctions and investigate the mechanism of translocation formation.
- To identify potential DNA recombination hotspots within the ETV6 and AML1 genes.
Main Methods:
- Gene mapping of the AML1 gene.
- Cloning of ETV6-AML1 fusion gene breakpoints from patient samples.
- Nucleotide sequence analysis of breakpoint junctions.
- Identification of insertion polymorphisms.
Main Results:
- The AML1 gene was localized between exons 1 and 3, with breakpoints predominantly found within AML1 intron 1.
- AML1 breakpoints were dispersed across intron 1, contrasting with the clustered ETV6 breakpoints.
- Sequence analysis revealed V(D)J consensus heptamer matches only on ETV6 alleles, suggesting V(D)J recombination is unlikely to be the sole mechanism.
- Several breakpoints and an insertion polymorphism were located near a polymorphic purine-pyrimidine tract in ETV6, indicating a potential recombination hotspot.
- An insertional polymorphism within the ETV6 bcr was identified, crucial for accurate genotype determination.
Conclusions:
- The dispersed nature of AML1 breakpoints and lack of consensus sequences on AML1 alleles challenge the hypothesis of simple V(D)J recombination in t(12;21) translocations.
- A polymorphic region in ETV6 intron 5 may predispose to DNA recombination events, including translocations and insertions.
- Accurate genetic analysis, particularly Southern blot interpretation, must account for insertional polymorphisms in the ETV6 bcr to prevent misdiagnosis.
Abstract:
The t(12;21)(p13;q22) translocation, fusing the ETV6 and AML1 genes, is the most frequent chromosomal translocation associated with pediatric B-cell precursor acute lymphoblastic leukemia. Although the genomic organization of the ETV6 gene and a breakpoint cluster region (bcr) in ETV6 intron 5 has been described, mapping of AML1 breakpoints has been hampered because of the large, hitherto unknown size of AML1 intron 1. Here, we report the mapping of the AML1 gene between exons 1 and 3, cloning of ETV6-AML1 breakpoints from different patients, and localization of the AML1 breakpoints within AML1 intron 1. In contrast to the tightly clustered ETV6 breakpoints, the AML1 breakpoints were found to be dispersed throughout AML1 intron 1. Although nucleotide sequence analysis of the breakpoint junctions showed several 5/7 matches for the V(D)J consensus heptamer recognition sequence, these matches were present only on the ETV6 alleles and not on the AML1 alleles, making it unlikely that the translocations were mediated by a simple V(D)J recombination mistake. Interestingly, several breakpoints as well as a stable insertion polymorphism mapped close to a polymorphic, alternating purine-pyrimidine tract in the ETV6 gene, suggesting that this region may be prone to DNA recombination events such as insertions or translocations. Finally, the presence of an insertional polymorphism within the ETV6 bcr must be recognized to avoid incorrect genotype designation based on Southern blot analysis.