Neuroblastoma Breakpoint Figure 6. Oncomine expression analysis of ACCN1 in neuroblastoma tumors. Significantly weaker ACCN1 expression is observed in tumors with MYCN amplification (MNA), in higher stage tumors (stage 3+4), and in tumors with 1p36 loss of heterozygosity (1p36 LOH), but not in tumors with 11q23 LOH. doi:10.1371/journal.pone.0002207.g006 Discussion Constitutional chromosomal abnormalities in patients bearing specific tumors have often been used to clone new TSGs. Here, we describe the breakpoint cloning of a constitutional translocation t(1;17)(p36.2;q11.2) present in a neuroblastoma patient. Aberra- tions in the chromosomal regions involved in this translocation are recurrent in neuroblastoma, and so we hypothesized that this translocation predisposed the patient to the development of neuroblastoma. We cloned both translocation breakpoints and identified on chromosome 1 a novel gene that is interrupted by the translocation. This gene is a member of the recently described NBPF gene family, which comprises 22 genes with highly repetitive exon types [27–29]. As these genes are located in regions of segmental duplications on chromosome 1, they show high sequence homology in both coding and non-coding regions. Unbalanced translocations between chromosomes 1 and 17 are common in neuroblastomas, but currently it is unclear whether there is a link between these somatic translocations and the constitutional balanced translocation described here. Only a few of the translocation breakpoints have been cloned [35,36], and no unifying underlying mechanisms for the translocations have been proposed. Two groups reported increased frequency of chromo- some 11q translocations in regions of segmental duplications [37,38]. In this respect, is striking that the chromosome 1 breakpoint in the t(1;17) translocation described here is also localized in a region of segmental duplication. NBPF1 is located on human chromosome 1p36. Though it is located outside of the smallest region of overlap defined by a large- scale analysis of neuroblastoma tumors [15], at least one copy of the region encompassing the NBPF1 gene is deleted in most neuroblastomas with 1p deletion. Tumor material from the neuroblastoma patient with the constitutional translocation was not available for genetic analysis, so we cannot exclude the possibility that the der(17) was lost in the tumor, leading to loss of the 1p segment distal to the translocation breakpoint. All genes located distal to the NBPF1 gene would therefore show LOH in the tumor, including a number of recently identified TSGs located in this region [13,14]. Alternatively, as our NBPF1 expression profiling revealed a decreased expression in neuroblastoma cell lines with a heterozygous deletion of the NBPF1 locus, it is also possible that NBPF1 acts as a haploinsufficient gene. The expression pattern of NBPF1 is reminiscent of the expression analysis of other genes located in the 1p35-36 region, where 15– 20% of the genes showed a lower expression level in neuroblas- toma tumors and cell lines with 1p loss of heterozygosity [17,39]. It has been proposed that the combined diminished expression of these genes, rather than the inactivation of one single classical TSG, could cause the unfavorable outcome associated with 1p deletions in neuroblastoma. In addition to analyzing specifically the NBPF1 gene, we also analyzed the complete NBPF gene family in neuroblastoma cell lines; most amplicons showed a significantly lower expression level in neuroblastoma cell lines with NBPF1 loss. In these cell lines the deletion was limited to NBPF1 and NBPF3, and the other NBPF members, located on chromosome 1q21, apparently cannot fully compensate for the decreased expression of the two deleted NBPF genes. Our qRT-PCR analyses were complemented by a meta-analysis using the Oncomine database [32]. These analyses showed decreased NBPF levels in neuroblas- tomas that relapsed within five years. During our previously reported analysis of the NBPF family, we detected structural variation in the NBPF1 gene in different individuals [27]. Similar results were reported in large-scale studies investigating copy- number variation between people [30]. These structural variants of NBPF1 and its paralogs should be taken into consideration when choosing amplicons and analyzing the expression levels of these genes in different cell lines. Furthermore, expression of NBPF1 in a human colorectal cell line severely suppressed soft agar colony formation, demonstrating PLoS ONE | www.plosone.org 9 May 2008 | Volume 3 | Issue 5 | e2207