Neuroblastoma Breakpoint Figure 3. Genomic overview of the translocation breakpoints of the der(1) and der(17) chromosomes. Schematic representation of the normal chromosome 1p36 and 17q12 regions is shown in the central box. On chromosome 1 (black bars) both translocation breakpoints are located in intronic regions of the NBPF1 gene, which is only partially shown (indicated by the dotted line). The breakpoint of the der(1) chromosome is located in a LINE repeat, shown as a black box labeled L1-PA4 on the genomic region. The black boxes below the genomic sequence represent the exons of the NBPF1 gene, with the exon type [27] indicated under each box. The translocation resulted in deletion of 5,215 bp. The translocation breakpoint on chromosome 17 (white bars) is located in an ACCN1 intron and disrupts isoform 2 but leaves isoform 1 intact. Transcription starts are indicated by angled arrows. The breakpoint is located in a LINE repeat, shown as a white box labelled L2 on the genomic region. White boxes below the genomic sequence represent the exons of the ACCN1 gene. On the der(1) chromosome (top), the NBPF1 gene is disrupted, giving rise to two chimeric cDNAs (shown as NBPF1-CHI, with the grey box representing the chromosome 17-derived fusion partners). The promoter region and first exon of ACCN1 isoform 2 are translocated to the der(1) chromosome, but no chimeric transcripts have been isolated yet (shown with the question mark and dotted line). On the der(17) chromosome (bottom), ACCN1 isoform 1 is unaffected by the translocation, but as the promoter and first exon of isoform 2 are translocated, isoform 2 is probably no longer expressed (shown by the dotted line and question mark). The last three NBPF1 exons (shown as exons of types 11, 12, and 14) are translocated to the der(17) chromosome. Arrows on the genomic sequence represent promoter sequences, and arrowheads in the transcripts show the orientation of the genes; tel, telomeric end; cen, centromeric end. The figure is not drawn to scale. doi:10.1371/journal.pone.0002207.g003 some primers can also bind to multiple positions on one transcript. We chose two amplicons that occur only once in a single NBPF transcript: on exon types 8-9 (amplicon B) and on the 39 UTR (amplicon D). In addition, we used two amplicons that can occur in multiple copies in the same transcript: one amplicon on the 59 repeat exons (amplicon A) and one amplicon on the 39 repeat exons (amplicon C). In the reference NBPF1 transcript (GenBank Acc Nu NM_017940), amplicon A is present in two copies and amplicon C as one copy (Figure 4A). However, in transcripts from other NBPF paralogs, amplicon A can be present as only one copy and amplicon C can be present as multiple copies [27]. These amplicons were analyzed in a panel of 31 neuroblastoma cell lines, some with a heterozygous deletion of NBPF1 (n = 25) and some with two copies of the NBPF1 gene (n = 5). As expected, the expression levels of amplicons B and D were comparable (Pearson correlation coefficient 0.95), but in some cases these levels differed considerably from those of amplicons A and C (Pearson correlation coefficient 0.86 for both A–D and C–D). All together, this analysis showed that the expression of all NBPF transcripts combined varied widely between different neuroblastoma cell lines: expression of amplicon C in cell lines SK-N-FI, SK-N-SH and NLF was up to fifteen-fold higher than in STA-NB-3. However, the expression level of amplicon B in these three cell lines was only six- to eleven-fold higher than in STA-NB-3. This difference was probably due to increased expression of transcripts containing multiple copies of amplicon C (e.g. NBPF10) [27] in the SK-N-FI, SK-N-SH and NLF cell lines as compared to STA-NB-3. For all amplicons, the median expression level of the NBPF family for cell lines with NBPF1 loss was lower than for cell lines with a normal 1p chromosome (Figure 4B). For amplicons A, B and D, the difference was statistically significant (p,0.05) but for amplicon C it was not (p = 0.13). Moreover, meta-analysis using the Oncomine database [32] revealed a significantly lower expression level of NBPF transcripts in neuroblastomas that relapsed within five years than in those that did not relapse (Figure 4C) [33]. The abovementioned primers amplified all NBPF transcripts, but the constitutional translocation disrupted only the NBPF1 PLoS ONE | www.plosone.org 6 May 2008 | Volume 3 | Issue 5 | e2207