Figure 2. Cloning of the constitutional t(1;17) translocation breakpoints in the cell hybrids 32-2F53VIII and 32-7A. Nucleotide sequences of the der(17) (A) and der(1) (B) regions flanking the translocation breakpoints. (C) Compilation of normal and derivative sequences at the breakpoint sites (NCBI Build 36.1) showing complete preservation of chromosome 17 sequences but not of chromosome 1 sequences. doi:10.1371/journal.pone.0002207.g002 Analysis of sequences flanking the constitutional t(1;17) translocation breakpoints Previous FISH experiments indicated that the constitutional translocation was balanced [7]. To verify this at the molecular level, we compared the chromosome 17 sequences obtained by GenomeWalker PCR to the sequence of the cosmid contig spanning the der(17) translocation breakpoint. We found that the translocation was indeed balanced on this level (Figure 2C). However, comparison of the chromosome 1 sequences to the human genome reference sequence (NCBI Build 36.1) revealed greater complexity. In both derivative chromosomes, a few nucleotides of unknown origin had been inserted between the chromosome 1 and chromosome 17 sequences (Figures 1 and 2), a feature frequently observed in reciprocal constitutional translocations [22]. Remark- ably, both the chromosome 1 translocation breakpoint cloned from the der(1) chromosome and the chromosome 17 breakpoint were located in LINE repeats (Figure 3). The repeat on chromosome 1 had been classified as type L1-PA4 [23], and the LINE repeat on chromosome 17 as type L2. However, when we compared these two LINE repeats, no significant sequence homology was found. On the other hand, the chromosome 1 breakpoint cloned from the der(17) chromosome was located in a repeat-free region. We previously reported that ACCN1 is the closest gene to the 17q breakpoint [18], but after completion of the human genome sequence it became clear that the translocation disrupts one of the isoforms of ACCN1 (Figure 3). This gene encodes an amiloride- sensitive cation channel [24] with two transmembrane domains [25]. It was shown that the ACCN1 gene is expressed as two isoforms differing in their 59UTR and N-terminal domain (Figure 3) [26]. All exons encoding isoform 1 (GenBank Acc Nu NM_183377.1) are located proximal to the translocation break- point, whereas the first exon of isoform 2 (GenBank Acc Nu NM_001094.4) is located distal to the breakpoint. The remaining exons of isoform 2 are shared with isoform 1 and are proximal to the breakpoint. The translocation thus disrupts isoform 2 of the ACCN1 gene without disturbing isoform 1 (Figure 3). Both isoforms are also found in mouse and rat, strengthening their functional relevance. On the translocated 1p-fragment of the der(17) Neuroblastoma Breakpoint chromosome, we identified three exons encoding the carboxy- terminal domain of the recently described NBPF1 protein. These are shown as exons of types 11, 12 and 14 in figures 1C and 3 (see [27] for numbering of NBPF1 exon types). NBPF1 is a member of a novel gene family with an intricate genomic structure [27–29]. Analysis of the der(1) sequences proximal to the breakpoint revealed the presence of two exons (shown as exons of types 6 and 7 in Figure 1D and Figure 3) that encode an internal domain of the NBPF1 protein. From this we conclude that the two breakpoints are located within distinct introns of the NBPF1 gene. Although the translocation appeared cytogenetically balanced, molecular cloning of the breakpoint sequences revealed a deletion of part of the NBPF1 gene. Recent reports describing structural variation in the human genome have shown recurrent structural abnormalities in the NBPF1 locus in normal individuals [30,31]; we also observed these abnormalities in our analysis of NBPF1 cDNA clones [27]. Due to this diversity, it is not possible to precisely determine the length of the deleted genomic fragment on chromosome 1 in this patient. However, compared to the human genome reference sequence (NCBI Build 36.1), a fragment comprising at least four exons of the NBPF1 gene is missing (5,215 bp nucleotides: 16,764,933 to 16,770,147 of chromosome 1). Chimeric transcripts resulting from the translocation Chromosomal translocations within a gene can generate one or two chimeric genes with altered functions. The two open reading frames disrupted in the t(1;17) translocation are encoded by complementary DNA strands, ruling out the possibility of chimeric NBPF1-ACCN1 transcripts (Figure 3). To isolate other chimeric cDNAs resulting from the t(1;17) translocation, we performed RACE (Rapid Amplification of cDNA Ends) analyses on the two hybrid cell lines, each of which contained one of the derivative chromosomes. We amplified two chimeric NBPF1 transcripts in the 32-7A cell line (Figure 3). In one chimeric transcript, the type-7 exon sequence was spliced to chromosome-17 sequences located 338 bp distal to the breakpoint, yielding a transcript extended by 295 nucleotides derived from chromosome 17, and an open reading frame extended by 34 additional codons (GenBank Acc No. AF420438). In a second chimeric transcript, the type-7 exon sequence was spliced to chromosome-17 sequences located 8,602 bp from the breakpoint, yielding a transcript extended by 325 nucleotides derived from chromosome 17, and an NBPF1 open reading frame extended by 11 additional codons (GenBank Acc Nu AF420439). The sequences derived from chromosome-17 were not found in other sequences known to be expressed, and were therefore classified as non-expressed sequences. An in silico analysis of the chimeric proteins encoded by these transcripts did not reveal any other identifiable domains. For ACCN1, we were unable to detect any chimeric cDNAs in the two hybrid cell lines containing the derivative chromosomes. Expression profiling in neuroblastoma cell lines and tumors A typical feature of a classical TSG is that its function is inactivated in different tumors. This inactivation can be caused by lack of expression due to deletion or promoter silencing. In a first quantitative RT-PCR assay we assessed four amplicons distributed across the NBPF transcripts (Figure 4A, top panel). This allowed us to define the expression profile of the complete NBPF gene family in neuroblastoma cell lines, as the primers we used bind to regions that show high sequence identity between the different NBPF paralogs. Additionally, due to the repetitive nature of the NBPF genes [27], PLoS ONE | www.plosone.org 5 May 2008 | Volume 3 | Issue 5 | e2207