Alu elements mediate MYB gene tandem duplication in human T-ALL

Abstract

Recent studies have demonstrated that the MYB oncogene is frequently duplicated in human T cell acute lymphoblastic leukemia (T-ALL). We find that the human MYB locus is flanked by 257-bp Alu repeats and that the duplication is mediated somatically by homologous recombination between the flanking Alu elements on sister chromatids. Nested long-range PCR analysis indicated a low frequency of homologous recombination leading to MYB tandem duplication in the peripheral blood mononuclear cells of approximately 50% of healthy individuals, none of whom had a MYB duplication in the germline. We conclude that Alu-mediated MYB tandem duplication occurs at low frequency during normal thymocyte development and is clonally selected during the molecular pathogenesis of human T-ALL.

Figure 1.

The MYB gene is tandemly duplicated in human T-ALL cell lines and patient samples. (A) Ideogram of chromosome 6 showing location of the MYB gene on q23. (B) Array CGH performed on DNAs from 17 T-ALL cell lines with Human Genome CGH 44K Microarrays (Agilent). A localized region on 6q23 surrounding the MYB locus is shown to have increased copy number in six of the cell lines. A 2.3-Mb region of chromosome 6 is shown. The top is centromeric, and the bottom is telomeric. Part of the AHI1 gene directly downstream of MYB is also amplified. NA indicates a probe in an intergenic region. Red indicates increased copy, blue indicates decreased copy, and the intensity of the color reflects the level of increase or decrease. (C) DNAs from leukemic cells in the diagnostic bone marrow of eight T-ALL patients were similarly analyzed by array CGH using Human Genome CGH 244K Microarrays, with an increased MYB copy number identified in two cases. A 500-kb region of chromosome 6 is shown. (D) Fiber-FISH on a T-ALL (TALL1) cell line with a diploid MYB copy number. The fosmid encompassing most of the MYB gene is labeled in green; a fosmid immediately 3′ of the MYB coding sequence is labeled in red. (E) Fiber-FISH on the Supt13 cell line showing a duplication of both fosmids spanning the entire MYB locus oriented in tandem on the same DNA fiber.

Figure 2.

MYB duplication is mediated by Alu elements on chromosome 6. (A) Detection of the MYB tandem duplication by long-range PCR. Primers were designed based on the fosmids used in the fiber-FISH experiments (Fig. S3). Primers 1, 6, and 7 were used in long-range PCR to detect the MYB duplication. Primer 2 is a control primer for long-range PCR. Primers 3, 4, 5, and 8 are nested primers used to generate a smaller product that was sequenced. Arrows inside boxes indicate Alu elements and their orientation. Insets show PCR results on representative cell lines. (left) Primers 1 and 6 were used in reaction 1; primers 1 and 7 were used in reaction 2; and primers 1 and 2 were used in reaction 3. (right) After three rounds of nested PCR, a band of 1 kb (reaction 3) was obtained in the CEM cell line, but not in normal thymocyte DNA. Primers 3 and 8 were used in reaction 1; primers 4 and 8 in reaction 2; and primers 5 and 8 in reaction 3. (B) Sequence in upper case is 116 kb downstream of MYB in the AHI1 locus. (C) Sequence in lower case is 3.5 kb upstream of MYB. (D) Sequences 1–5 represent the 5 different junctions between the 2 copies of MYB detected in T-ALL cell lines and primary samples. One additional breakpoint (sequence 6) was identified in normal lymphoblastoid cell line DNA. Underlined sequences represent the proposed crossover region. Fig. S3 is available at http://www.jem.org/cgi/content/full/jem.20071637/DC1.

Figure 3.

Proposed mechanism of MYB tandem duplication. (A) A DNA double-strand break occurs in (or near) the Alu element downstream of MYB. (B) The broken DNA strand invades the sister chromatid and pairs with the Alu element upstream of MYB. (C) DNA repair synthesis continues through the MYB gene, and the newly synthesized strand anneals to the broken chromatid by strand annealing. (D) The result is duplication of the sequence between the Alu elements. The sequence of the Alu element between the two copies of MYB (gray box) is a hybrid of the two repeats on either side of MYB, depending on where the strand anneals.