Chromosome 12p deletions in TEL-AML1 childhood acute lymphoblastic leukemia are associated with retrotransposon elements and occur postnatally

Abstract

TEL-AML1 (ETV6-RUNX1) is the most common translocation in the childhood leukemias, and is a prenatal mutation in most children. This translocation has been detected at a high rate among newborns ( approximately 1%); therefore, the rate-limiting event for leukemia seems to be secondary mutations. One such frequent mutation in this subtype is partial deletion of chromosome 12p, trans from the translocation. Nine del(12p) breakpoints within six leukemia cases were sequenced to explore the etiology of this genetic event, and most involved cryptic sterile translocations. Twelve of 18 del(12p) parent sequences involved in these breakpoints were located in repeat regions (8 of these in long interspersed nuclear elements). This stands in contrast with TEL-AML1, in which only 21 of 110 previously assessed breakpoints (19%) occur in DNA repeats (P=0.0001). An exploratory assessment of archived neonatal blood cards revealed significantly more long interspersed nuclear element CpG methylations in individuals at birth who were later diagnosed with TEL-AML1 leukemia, compared with individuals who did not contract leukemia (P=0.01). Nontemplate nucleotides were also more frequent in del(12p) than in TEL-AML1 junctions (P=0.004), suggesting formation by terminal deoxynucleotidyl transferase. Assessment of six archived neonatal blood cards indicated that no del(12p) rearrangements backtracked to birth, although two of these patients were previously positive for TEL-AML1 using the same assay with comparable sensitivity. These data are compatible with a two-stage natural history: TEL-AML1 occurs prenatally, and del(12p) occurs postnatally in more mature cells with a structure that suggests the involvement of retrotransposon instability.

Figures

Figure 1

A. Array comparative genomic hybridization of chromosome 12, p-arm. Isothermal tiling path array with 390,000 probes spaced at 50 bp intervals. Log2 fluorescence ratios on Y-axis, tumor DNA (vertical up) vs. normal control (vertical down) on the Y-axis, the red line indicated the change point analysis. B. Smallest commonly deleted region on chromosome 12 from the 10 patients displayed in Figure 1. The identities of the genes located within this region are indicated at the right.

Figure 2

Structure of del(12p) breakpoints and translocations. A and B indicate patients #5 and 2, respectively, as an example of an interstitial breakpoint (patient #5) and translocation (#2). X-axis scales of CGH plots are indicated at the left of each plot. The breakpoint is shown, with arrows indicating the direction of the sequence as numbered in human genome. Position of primers for sequencing the breakpoint (diagrammatically, not to scale) are shown — inverse primers for patient 2, and LD-PCR for patient #5. The first round primers are indicated with lower case “a” and the second round primers with lower case “b”. See Wiemels et al., (Ref #32) for more detailed methodology. Position of the backtracking PCR is noted at the bottom.

Figure 2

Structure of del(12p) breakpoints and translocations. A and B indicate patients #5 and 2, respectively, as an example of an interstitial breakpoint (patient #5) and translocation (#2). X-axis scales of CGH plots are indicated at the left of each plot. The breakpoint is shown, with arrows indicating the direction of the sequence as numbered in human genome. Position of primers for sequencing the breakpoint (diagrammatically, not to scale) are shown — inverse primers for patient 2, and LD-PCR for patient #5. The first round primers are indicated with lower case “a” and the second round primers with lower case “b”. See Wiemels et al., (Ref #32) for more detailed methodology. Position of the backtracking PCR is noted at the bottom.

Figure 3

Electrophoretic gels of backtracking del(12p) experiments in five patients as an example of results. A. Secondary PCR for patient 1-F1 and 1-R1 primers. Lane 1 & 18, 1 kb ladder; lane 2,3,4,5,6,7: 100 to 0.001 ng patient 1 bone marrow serial dilution DNA; lane 8, 9, 10 & 17, blank; lanes 11,12, 14, and 15 various control lymphocyte DNAs from healthy NCCLS study control participants; and lane 13 & 16, 50 ng of patient #1 ANB card DNA. B. Secondary PCR reaction for patient 5. Primers: patient 5-F1 and R1. Lanes 1 & 18, 1 kb ladder; lane 2, 3, 5, 6, 7, 8, 9: 100, 10, 1, 0.1, 0.01, and 0.001 ng patient 5 bone marrow DNA; lanes 4, 10, 11, blank; lanes 12, 13, 15, and 16, various control DNAs from healthy controls; and lanes 14 & 17, 50 ng of patient 5 ANB card DNA. C, D, and E. patients #7, 8, and 10 respectively. Lanes 2-7: 100 to 0.001 ng patient 1 bone marrow serial dilution DNA in; lane 7 blank; lane 8 and 9, DNA from healthy controls; lane 10, DNA from a control ANB card; and lane 11, 50 ng DNA from the interrogated patient's ANB card. In C, the primary PCR reaction is shown along with the secondary reaction. Gels A and B were performed by a different technician than gels C-E.

Figure 4

Schematic representation showing the proximity and identity of repetitive elements in relation to the patient breakpoints. Alu sequences are in black, LINE elements in cross-hatched rectangles, and long terminal repeat sequences are open rectangles. The breakpoints are listed (a, b, c. . .) from left to right in relationship to the chromosome 12 arm (see Supplementary Table 2 for the data that this figure is based on).

Figure 5

LINE1-M1 methylation status in ANB cards from children who contracted leukemia later in their lives with TEL-AML1 translocations, other cALL diagnoses, and who did not contract leukemia. The controls are age, gender, and ethnicity frequency-matched to cases. Data was Log10 transformed to approximate normal distribution with the center box displaying two quartiles (bisected by the median) and the whiskers the marginal quartiles.