Cloning of AML1-LAF4 Fusion Gene in Childhood T-Cell Acute Lymphoblastic Leukemia with t(2;21)(q11;q22) by Bubble PCR Method for cDNA.

The AML1/RUNX1 gene is frequently rearranged by chromosomal translocations in acute leukemia. To date, more than 10 fusion genes involving AML1 have been cloned, such as AML1-MTG8 in acute myeloid leukemia (AML) with t(8;21), AML1-EVI1/MDS1 in therapy-related AML/myelodysplastic syndrome with t(3;21), and TEL/ETV6-AML1 in B precursor ALL with t(12;21). We analyzed a pediatric patient having T-cell acute lymphoblastic leukemia (T-ALL) with t(2;21)(q11;q22), and identified that the LAF4 gene on 2q11.2–12 was fused to the AML1 gene on 21q22 using the bubble PCR method for cDNA. The genomic breakpoints were within intron 7 of AML1 and intron 7 of LAF4 resulting in the in-frame fusion of exon 7 of AML1 and exon 8 of LAF4. LAF4 gene is a member of the AF4/FMR2 family, and was previously identified as a fusion partner of MLL in B-precursor ALL with t(2;11)(q11;q23), although AML1-LAF4 was in T-ALL. LAF4 is the first gene fused with both AML1 and MLL in acute leukemia. These findings provide new insights into the common mechanism of AML1 and MLL fusion proteins in the pathogenesis of ALL. In this study, we first applied the panhandle PCR method that is usually used for cloning the fusion partners of MLL or NUP98; however, no fusion transcripts could be obtained. Therefore, we searched for another method for cloning the fusion transcripts and successfully adapted the bubble PCR method for cloning the novel AML1-LAF4 fusion transcript. To date, bubble PCR has been performed for cloning unknown genomic fusion points but not fusion cDNAs. Using double-strand cDNA, we could apply the bubble PCR method for cloning fusion cDNA with rare non-specific products. Bubble PCR for cDNA could amplify in both 5′ to 3′ and 3′ to 5′ directions of the gene or transcript and handle any exons fused to unknown partners for amplification easily. This method will contribute to identifying numerous novel translocation partners more easily, and these findings may help to clarify the role of the fusion proteins in leukemogenesis.