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

Thorell K, Bergman A, Carén H, Nilsson S, Kogner P, Martinsson T, Abel F - BMC Med Genomics (2009)

Bottom Line: 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.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.Through two steps of verification, seven transcripts were found to significantly discriminate between favourable and unfavourable NB tumours.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical Genetics, Gothenburg University, S-405 30 Gothenburg, Sweden. kaisa.thorell@wlab.gu.se

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.

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: DNA sequencing analysis of the gene POU4F2 [GenBank: NM_004575] was performed on four favourable and four unfavourable primary tumours showing the lowest and highest expression values in their respective group (Table 1). Sixteen PCR-primer pairs covering the promoter region and the coding regions of POU4F2 were designed using Exon Primer and purchased from Invitrogen (Invitrogen, Carlsbad, CA). Of these, four pairs span the 5'UTR/promoter region including one covering the Wilms' tumour transcription factor (Wt1) binding site located at -1387 to -1377 from translation start (Figure 2) [28]. Exon 1 was covered by three amplicons and exon 2 of nine amplicons (Figure 2). Primer sequences are available on request. Touch down (TD) PCR was performed in 10 μl reactions containing 1× Coral Load PCR Buffer (Qiagen), 20 mM dNTP mix, 1× Q-solution (Qiagen), 0,25 U Hot Star TaqPlus DNA polymerase (Qiagen), 10 μM of forward (FWD) and reverse (REV) primer, respectively, and 50 ng of tumour DNA. The TD PCR program was optimized for GC-rich fragments and run at 95°C for 15 min before cycling 20 rounds of 98°C for 30 sec, 60°C for 30 sec (decreasing 0,5°C in every cycle), and 72°C for one minute – followed by 25 cycles of 98°C for 30 sec, 50°C for 30 sec and 72°C for 1 min and finally a 72°C extension step for 7 minutes. Amplification products were analysed for specificity on a 2% agarose gel before they were purified using AMPure magnetic beads (Agencourt Bioscience Corporation, Beverly, MA) using the Biomek NX pipetting robot (Beckman Coulter) and eluted in dH2O. Sequencing PCR was performed using the BigDye Terminator (BDT) v3.1 Cycle Sequence Kit (Applied Biosystems) in 10 μl reactions containing 6 μl 1:3 diluted PCR-template DNA, 1 μl BDT, 1× BDT buffer and 1,6 μM of the PCR primer, either FWD or REV. Sequence PCR was run under following conditions; 94°C for 3 min, followed by 50 cycles of 96°C for 30 sec, 50°C for 10 sec and 60°C for 3 min each. Sequencing products were cleaned using CleanSeq magnetic beads (Agencourt) using the Biomek NX and resuspended in 10 μl of High Dye formamide (Applied Biosystems). The sequencing products were separated with gel electrophoresis on the 3730 DNA analyser (Applied Biosystems) and the output data were viewed and analysed using softwares Sequencing Analysis v 5.0 and SeqScape v 2.5, both from Applied Biosystems. All eight primary tumours and the positive reference CEPH DNA (CEPH1347-02, Applied Biosystems) were successfully sequenced. Each finding was validated by a second PCR and sequencing reaction.


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

Thorell K, Bergman A, Carén H, Nilsson S, Kogner P, Martinsson T, Abel F - BMC Med Genomics (2009)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2743704&req=5

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.
Mentions: DNA sequencing analysis of the gene POU4F2 [GenBank: NM_004575] was performed on four favourable and four unfavourable primary tumours showing the lowest and highest expression values in their respective group (Table 1). Sixteen PCR-primer pairs covering the promoter region and the coding regions of POU4F2 were designed using Exon Primer and purchased from Invitrogen (Invitrogen, Carlsbad, CA). Of these, four pairs span the 5'UTR/promoter region including one covering the Wilms' tumour transcription factor (Wt1) binding site located at -1387 to -1377 from translation start (Figure 2) [28]. Exon 1 was covered by three amplicons and exon 2 of nine amplicons (Figure 2). Primer sequences are available on request. Touch down (TD) PCR was performed in 10 μl reactions containing 1× Coral Load PCR Buffer (Qiagen), 20 mM dNTP mix, 1× Q-solution (Qiagen), 0,25 U Hot Star TaqPlus DNA polymerase (Qiagen), 10 μM of forward (FWD) and reverse (REV) primer, respectively, and 50 ng of tumour DNA. The TD PCR program was optimized for GC-rich fragments and run at 95°C for 15 min before cycling 20 rounds of 98°C for 30 sec, 60°C for 30 sec (decreasing 0,5°C in every cycle), and 72°C for one minute – followed by 25 cycles of 98°C for 30 sec, 50°C for 30 sec and 72°C for 1 min and finally a 72°C extension step for 7 minutes. Amplification products were analysed for specificity on a 2% agarose gel before they were purified using AMPure magnetic beads (Agencourt Bioscience Corporation, Beverly, MA) using the Biomek NX pipetting robot (Beckman Coulter) and eluted in dH2O. Sequencing PCR was performed using the BigDye Terminator (BDT) v3.1 Cycle Sequence Kit (Applied Biosystems) in 10 μl reactions containing 6 μl 1:3 diluted PCR-template DNA, 1 μl BDT, 1× BDT buffer and 1,6 μM of the PCR primer, either FWD or REV. Sequence PCR was run under following conditions; 94°C for 3 min, followed by 50 cycles of 96°C for 30 sec, 50°C for 10 sec and 60°C for 3 min each. Sequencing products were cleaned using CleanSeq magnetic beads (Agencourt) using the Biomek NX and resuspended in 10 μl of High Dye formamide (Applied Biosystems). The sequencing products were separated with gel electrophoresis on the 3730 DNA analyser (Applied Biosystems) and the output data were viewed and analysed using softwares Sequencing Analysis v 5.0 and SeqScape v 2.5, both from Applied Biosystems. All eight primary tumours and the positive reference CEPH DNA (CEPH1347-02, Applied Biosystems) were successfully sequenced. Each finding was validated by a second PCR and sequencing reaction.

Bottom Line: 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.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.Through two steps of verification, seven transcripts were found to significantly discriminate between favourable and unfavourable NB tumours.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical Genetics, Gothenburg University, S-405 30 Gothenburg, Sweden. kaisa.thorell@wlab.gu.se

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.

No MeSH data available.


Related in: MedlinePlus