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Figure 9.3

Expression of genes near MYCN in samples showing amplification or over-expression of this gene. Map is derived from the UCSC genome database (http:// genome.ucsc.edu/cgi-bin/ hgGateway). Gray boxes indicate expression at least one standard deviation above the mean for that gene in tumor samples

1985; Ma et al. 1993). Oligonucleotide arrays have been used to monitor the effects of MYCN on gene expression in NB through analysis of human tumors and cell lines (Alaminos et al. 2003). An interesting finding that has been noted in previous studies (Nisen et al. 1988; Slave et al. 1990) is that MYCN mRNA expression levels did not always coincide with MYCN gene copy number. This implies that over-expression of MYCN occurs in some cases of NB without gene amplification and is probably due to alterations in transcriptional regulation. The clinical significance of over-expression in the absence of amplification is uncertain; however, in array studies, tumors with high levels of MYCN expression in the absence of amplification tend to cluster with amplified tumors with increased expression, demonstrating a correlation between level of MYCN and overall gene expression profile (Alaminos et al. 2003).

Alaminos and co-workers compared the expression profiles of tumors with and without high levels of MYCN mRNA using relatively stringent criteria and found that 222 of 62,839 probe sets identified genes with significant differential expression (Alaminos et al. 2003). Seventy-four probe sets detected genes that were up regulated and 148 that were down regulated in tumors with high levels of MYCN RNA. Some of these were believed to be direct targets of this oncogene based on altered expression in cell lines with induced expression of MYCN. The functional aspects of some of these genes included transcriptional regulators (HTATIP, HTATIP2, DDX1, MI-ER1 and NCYM), oncogenes (NCYM, RAB20), cell proliferation (CDCA7, CENPE, CDC2L2, PC-TAIRE2BP), and neural differentiation (HOXC10, PTN, FMNL, DNER, CLU, GDA, NRCAM, ECEL1 and SNPH), and correlate well with the lack of differentiation and high mitotic-karyorrhectic index which is common for MYCN amplified tumors. An expression map of the region corresponding to NB with high levels of MYCN expression (Fig. 9.3) demonstrated that MYCN is the only gene consistently expressed in all NB with 2p amplification and agrees with the findings of others (George et al. 1996; Hiemstra et al. 1994). The significance of over-expression of co-amplified genes is unknown.

A separate study investigating the regulation of gene expression by MYCN in a NB cell line using SAGE analysis had a very different result (Boon et al. 2001). In that study the majority of genes that were upregulated by MYCN were associated with ribo-some assembly and activity. Potential reasons for the differences between the findings in these studies include the over-representation of highly expressed genes in SAGE analysis, the different experimental systems (cell lines vs tumor samples), and the analytical methods used to identify differentially expressed

Figure 9.4

A positional gene expression map of 12q13-15 from 56416679 bp to 76539024 bp.Map is derived from the UCSC genome database (http://genome.ucsc.edu/cgi-bin/hgGateway). Each column is a single NB sample and each row is expression of an individual gene. Red highlight indicates expression level greater than two standard deviations above the mean.The five cases with obvious over-expression of this region are in the first five columns on the left.Note that the genes separate into two distinct regions, 12q13.3 and 12q15

genes. These disparate results emphasize the importance of technical and analytical factors in high-throughput molecular studies.

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