Different molecular mechanisms causing 9p21 deletions in acute lymphoblastic leukemia of childhood

Abstract

Deletion of chromosome 9p21 is a crucial event for the development of several cancers including acute lymphoblastic leukemia (ALL). Double strand breaks (DSBs) triggering 9p21 deletions in ALL have been reported to occur at a few defined sites by illegitimate action of the V(D)J recombination activating protein complex. We have cloned 23 breakpoint junctions for a total of 46 breakpoints in 17 childhood ALL (9 B- and 8 T-lineages) showing different size deletions at one or both homologous chromosomes 9 to investigate which particular sequences make the region susceptible to interstitial deletion. We found that half of 9p21 deletion breakpoints were mediated by ectopic V(D)J recombination mechanisms whereas the remaining half were associated to repeated sequences, including some with potential for non-B DNA structure formation. Other mechanisms, such as microhomology-mediated repair, that are common in other cancers, play only a very minor role in ALL. Nucleotide insertions at breakpoint junctions and microinversions flanking the breakpoints have been detected at 20/23 and 2/23 breakpoint junctions, respectively, both in the presence of recombination signal sequence (RSS)-like sequences and of other unspecific sequences. The majority of breakpoints were unique except for two cases, both T-ALL, showing identical deletions. Four of the 46 breakpoints coincide with those reported in other cases, thus confirming the presence of recurrent deletion hotspots. Among the six cases with heterozygous 9p deletions, we found that the remaining CDKN2A and CDKN2B alleles were hypermethylated at CpG islands.

Fig. 1

An example of custom array-CGH results: case 217-07. a Chromosome 9 profile with 9p21 deletion indicated by the box, b enlargement of the 9p21 region showing a homozygous deletion, c further enlargement of the locus under study; the arrows point to the edge between normal and deleted oligomeres

Fig. 2

a Graphic representation of the 9p21.3 deletions in all 18 cases. Grey lines represent the deleted region of every allele of each case. Overlap of two lines for the same case indicates a homozygous deletion. b Detailed map of the MTAP/CDKN2A/CDKN2B region. The ten cases whit homozygous/heterozygous deletions, whose breakpoints (at least one) map between 21,800,000 and 22,100,000 bp, are reported

Fig. 3

MS-PCR analysis for CDKN2A and CDKN2B showing the products from wild-type/unmodified DNA (W), unmethylated bisulfite-modified DNA (U) and methylated bisulfite-modified DNA (M). On the left: molecular weight marker (GelPilot 50 bp). a, b, and c MS-PCR products using specific primers for p16INK4a, p14ARF and p15INKb, respectively, from case 1623-06