Verification of genes differentially expressed in neuroblastoma tumours: a study of potential tumour suppressor genes

Abstract

Background: One of the most striking features of the childhood malignancy neuroblastoma (NB) is its clinical heterogeneity. Although there is a great need for better clinical and biological markers to distinguish between tumours with different severity and to improve treatment, no clear-cut prognostic factors have been found. Also, no major NB tumour suppressor genes have been identified.

Methods: In this study we performed expression analysis by quantitative real-time PCR (QPCR) on primary NB tumours divided into two groups, of favourable and unfavourable outcome respectively. Candidate genes were selected on basis of lower expression in unfavourable tumour types compared to favourables in our microarray expression analysis. Selected genes were studied in two steps: (1) using TaqMan Low Density Arrays (TLDA) targeting 89 genes on a set of 12 NB tumour samples, and (2) 12 genes were selected from the TLDA analysis for verification using individual TaqMan assays in a new set of 13 NB tumour samples.

Results: By TLDA analysis, 81 out of 87 genes were found to be significantly differentially expressed between groups, of which 14 have previously been reported as having an altered gene expression in NB. In the second verification round, seven out of 12 transcripts showed significantly lower expression in unfavourable NB tumours, ATBF1, CACNA2D3, CNTNAP2, FUSIP1, GNB1, SLC35E2, and TFAP2B. The gene that showed the highest fold change in the TLDA analysis, POU4F2, was investigated for epigenetic changes (CpG methylation) and mutations in order to explore the cause of the differential expression. Moreover, the fragile site gene CNTNAP2 that showed the largest fold change in verification group 2 was investigated for structural aberrations by copy number analysis. However, the analyses of POU4F2 and CNTNAP2 showed no genetic alterations that could explain a lower expression in unfavourable NB tumours.

Conclusion: Through two steps of verification, seven transcripts were found to significantly discriminate between favourable and unfavourable NB tumours. Four of the transcripts, CACNA2D3, GNB1, SLC35E2, and TFAP2B, have been observed in previous microarray studies, and are in this study independently verified. Our results suggest these transcripts to be markers of malignancy, which could have a potential usefulness in the clinic.

Figure 1

Schematic representation of the study approach. Verification group1: Eighty-nine genes were selected for gene expression analysis using TaqMan Low Density Array, TLDA, see text for details. In the TLDA analysis, six favourable (F) and six unfavourable (UF) tumours were included. Verification group 2: Twelve candidate genes were selected for validation by TaqMan individual QPCR, TM (see Table 3 for selection criteria). A new, randomly selected set of favourable (n = 7) and unfavourable (n= 6) tumours were used in the analyses, and all samples were run in duplicates (×2).

Figure 2

Schematic representation of the POU4F2 gene. Green lines represent exons where the green boxes specify the protein coding parts. Position 1 marks the translation start. The upper grey lines represent the fragments amplified by selected primer pairs. The lower dark blue box indicates predicted CpG islands and the upper blue line marks the region covered by methylation analysis.

Figure 3

Fold scatter plot of 12 transcripts studied by both TLDA and TaqMan. The geometric means of the relative expression in favourable tumours of verification group 2 are used as reference (Fold = 1). Open squares = technical replicate group studied by TLDA but also represented on the microarray; Open circles = verification group 1 studied only with TLDA; Filled triangles = verification group 2 studied by individual TaqMan assays. The fold change (FC) between groups is based on expression values in verification group 2. Group: F = Favourable tumour types: Group UF = Unfavourable tumour types.

Figure 4

BSP sequencing of the POU4F2 promoter. Cell-line SK-N-BE shows methylation of CpG sites in the POU4F2 promoter. Cytosines (blue peaks) marked with arrows, are modified into thymidines (red peaks) if not methylated. Cell-line SK-N-BE is compared to unmethylated primary tumour 9R9.