Open Access Highly Accessed Research article

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

Kaisa Thorell12, Annika Bergman3, Helena Carén1, Staffan Nilsson2, Per Kogner4, Tommy Martinsson1 and Frida Abel12*

Author Affiliations

1 Department of Clinical Genetics, Gothenburg University, S-405 30 Gothenburg, Sweden

2 Department of Mathematical Statistics, Chalmers University of Technology, S-412 96 Gothenburg, Sweden

3 Department of Pathology, Lundberg Laboratory for Cancer Research, SU/Sahlgrenska, S-413 45, Sweden

4 Childhood Cancer Research Unit, Karolinska Institute, Astrid Lindgren Children's Hospital Q6:05, S-171 76 Stockholm, Sweden

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BMC Medical Genomics 2009, 2:53  doi:10.1186/1755-8794-2-53

Published: 17 August 2009

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.