Oncogenomics WAVE3, an actin-polymerization gene, is truncated and inactivated as a result of a constitutional t(1;13)(q21;q12) chromosome translocation in a patient with ganglioneuroblastoma Khalid Sossey-Alaoui1, Guanfang Su1, Eda Malaj2, Bruce Roe2 and John K Cowell1 1Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York, USA2Department of Chemistry, University of Oklahoma, Norman, Oklahoma, USA Correspondence to: J K Cowell, Department of Cancer Genetics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA; E-mail: John.Cowell@RoswellPark.org Abstract Neuroblastoma (Nb) is a malignancy of the sympathetic nervous system which affects children in their first decade. It is the most common extra-cranial solid tumor in children with an incidence of approximately 1 in 8-10 000 live births annually and accounts for approximately 10% of all children's cancers. Ganglioneuroblastoma is a relatively benign form of Nb and consists of a mixture of fibrils, mature and maturing ganglion cells, as well as undifferentiated neuroblasts. During routine cytogenetic analysis of patients with different manifestations of neuroblastoma we have identified one patient with ganglioneuroblastoma that carries an apparently balanced t(1:13)(q21:q12) reciprocal translocation. Positional cloning of the translocation breakpoint on chromosome 13 resulted in the mapping of the breakpoint between coding exon 2 and exon 3 of WAVE3, a member of WASP gene family. Although the breakpoint region on chromosome 1 was localized to within 2 kb of genomic sequence, no gene was found to be interrupted on this chromosome. The WAVE3 transcript is mainly expressed in the nervous system and, like all the members of the WASP gene family, WAVE3 is a key element in actin polymerization and cytoskeleton organization. WAVE3, therefore, is important for cell differentiation and motility and its expression is lost in a number of low grade and stage 4S tumors. From analysis of its expression pattern and function, WAVE3 is a candidate tumor suppressor gene, at least in some forms of neuroblastoma. Oncogene(2002) 21, 5967-5974. doi:10.1038/sj.onc.1205734 Keywords WAVE3; neuroblastoma; actin polymerization Introduction Neuroblastoma (Nb) is the most common extracranial solid tumor in childhood, accounting for 8% of all pediatric malignancy. Family studies (Kushner et al., 1986) and epidemiological analyses (Knudson and Strong, 1972) suggests that the Nb tumor phenotype segregates as an autosomal dominant trait with reduced (63%) penetrance. This led to the proposal that tumorigenesis was a consequence of a loss of function of a critical gene(s) in embryonic tumor cells in a way similar to that proposed for retinoblastoma (Knudson, 1971). However, because Nb families are rare (Kushner et al., 1986), it has not yet been possible to assign any Nb predisposition gene to a particular chromosome region using conventional linkage studies. This analysis may be further complicated by the possibility that Nb may, in fact, represent several different clinical entities. Thus, ganglioneuroblastoma, which is considered a relatively benign tumor, consists of both immature neuroblasts as well as fully differentiated neuronal cells. Ganglioneuroma, on the other hand, consists entirely of differentiated cells. What is not known is whether these two variants represent distinct diseases or whether ganglioneuroma arises as a result of differentiation of cells from a ganglioneuroblastoma. The most aggressive forms of Nb within the disease spectrum consist of undifferentiated neuroblasts. It has been consistently demonstrated that undifferentiated Nb cells can differentiate in vitro when exposed to a variety of inducing agents (Ponzoni et al., 1991; Lanciotti et al., 1992), indicating that differentiation signals in these cells can overcome the malignant phenotype. Thus, whether neuroblastoma represents the end point or a continuous progression from the more benign forms, or whether they are different diseases, is not clear. The indication that Nb in fact probably represents several different diseases has been suggested from survival studies (Castleberry et al., 1999). The final conundrum comes from the special form of the disease called 'stage 4S'. This tumor presents as a low-grade primary tumor in children under the age of 12 months, but is associated with distant metastases primarily to the liver and bone marrow. The feature that makes this form of Nb stand out is the fact that these tumors can spontaneously resolve themselves (D'Angio et al., 1971). Clearly, in this sub-type, there has been a massive expansion of primitive neuroblasts, although these cells can still respond to differentiation or apoptotic signals. Again whether this represents a distinct form of the disease or arises through a completely different pathway has not been determined. Analysis of constitutional chromosome abnormalities in individuals who develop particular tumor types has frequently indicated the site of predisposition gene. This has been the case for retinoblastoma (Yunis and Ramsay, 1978), Wilms' tumor (Riccardi et al., 1978), neurofibromatosis (Fountain et al., 1989; O'Connell et al., 1989), and familial adenomatous polyposis (Varesco et al., 1989). These structural abnormalities are predominantly deletions, but where translocations are identified, the breakpoints usually interrupt the predisposition gene (reviewed in Mitchell, 1991). A few cases of Nb with constitutional chromosome abnormalities have been described (Anderson et al., 2001; Fryns, 1996; Laureys et al., 1990, Nagano et al., 1980; Sanger et al., 1984). In particular, two patients with stage 3 and 4 tumors were shown to carry constitutional chromosome changes involving 1p35-36. Laureys et al. (1990) reported a patient with a constitutional reciprocal translocation which was later defined (van Roy et al., 1997) although no genes have been reported to be involved in this rearrangement to date. We (Mead and Cowell, 1995) described a patient with stage 4S disease, and a constitutional t(1;10)(p22;q21) translocation. The chromosome 1 translocation breakpoint does not lie within the consensus LOH region seen in advanced stage tumors but does lie in the LOH region reported for stage 4S tumors (Mora et al., 2000). Cloning the breakpoint from this translocation identified an in-frame fusion of two genes resulting in the truncation and inactivation of the NB4S/EVI5 gene (Roberts et al., 1998a,b). The association of different chromosome rearrangements with different subtypes of the disease was further demonstrated in the report by Michalski et al. (1992) who described a patient with ganglioneuroblastoma and a constitutional t(1;13)(q23;q12) reciprocal chromosome translocation. The breakpoint on chromosome 1 was in a different position compared with the patients with stage 3, 4 and 4S. Recent reports suggest that amplification of this region is frequently seen in low-grade tumors and so may represent a subtype-specific gene locus for Nb (Hirai et al., 1999). We have now cloned the breakpoints associated with this 1;13 translocation and shown that WAVE3 on 13q12, which is though to be involved in actin-polymerization (Suetsugu et al., 1999) is physically interrupted and inactivated as a result of this translocation event. We have shown that the WAVE3 transcript is down regulated in three sporadic neuroblastoma tumors. No known or predicted gene is associated with the 1q23 breakpoint. Results Mapping the breakpoint on chromosome 1 To study the de novo constitutional t(1;13)(q22;q12) translocation in patient DG a somatic cell hybrid, DGF27 (Michalski and Cowell, 1993), was isolated which retained the derivative chromosome 1, (1pter-q21;13q12-qter).
