Background: MYCN amplification is frequently found in high-risk NBs. Amplification is usually manifested cytogenetically by extrachromosomal double minutes (DMs) or by chromosomally integrated homogenously-staining regions (HSRs). MYCN is the only gene that is consistently amplified in NB tumors and cell lines. However, little is known about the structure of MYCN amplicons or the mechanisms that give rise to MYCN amplification. Here, we analyzed the NB lines Kelly, BE2C and LA-N-5 to determine the DNA sequence of amplicon junctions.
Methods: We performed SNP-Arrays on 27 NB cell lines and used this information to construct a map of amplicons. Real-time PCR was employed to identify the boundaries of each amplified region. Once the boundaries of each amplicon end were narrowed to <3 kb, PCR and DNA sequencing were performed to determine the exact sequences of amplication junctions to determine the orientation of single or adjacent amplicons (head-tail, head-head, tail-tail).
Results: All amplicons analyzed were composed of a simple repeat of genomic DNA fragments: Kelly (chr2:15,834,141-16,840,864 ~1Mbp-HSR), SK-N-BE2C (chr2:16,076,406-16,602,741 ~0.53Mbp/HSR), LA-N-5 (chr2:15,489,976-17,114,868 ~1.7Mbp/DMs), and a small fragment (241 bp) in the same orientation (head to tail).　The MYCN gene (chr2:16,082,046-16,087,129) was included in all the amplicons studied. None of the amplicon junctions defined at the nucleotide level had either novel, repeat or rearranged sequences. However, we found imperfect hairpin structures around the amplicon junctions in two NB lines.
Conclusion: Our results suggest that the junctional sequences of MYCN amplicons are a perfect match to the genomic DNA sequence, with no gain, loss or rearrangement of sequences at the junctions. This indicates that there may be a different mechanism responsible for DNA amplification in human NBs other than the well accepted break-bridge repair model.