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Exon expression arrays as a tool to identify new cancer genes.

Schutte M, Elstrodt F, Bralten LB, Nagel JH, Duijm E, Hollestelle A, Vuerhard MJ, Wasielewski M, Peeters JK, van der Spek P, Sillevis Smitt PA, French PJ - PLoS ONE (2007)

Bottom Line: An estimated 10-20% of cancer-related gene mutations result in skipping of one or more exons in the encoded transcripts.PAC reduced the number of candidate genes/exons for subsequent mutational analysis by two to three orders of magnitude and had a substantial true positive rate.Importantly, of 112 randomly selected outlier exons, sequence analysis identified two novel exon skipping events, two novel base changes and 21 previously reported base changes (SNPs).

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Josephine Nefkens Institute, Erasmus University Medical Center, Rotterdam, The Netherlands. a.schutte@erasmusmc.nl

ABSTRACT

Background: Identification of genes that are causally implicated in oncogenesis is a major goal in cancer research. An estimated 10-20% of cancer-related gene mutations result in skipping of one or more exons in the encoded transcripts. Here we report on a strategy to screen in a global fashion for such exon-skipping events using PAttern based Correlation (PAC). The PAC algorithm has been used previously to identify differentially expressed splice variants between two predefined subgroups. As genetic changes in cancer are sample specific, we tested the ability of PAC to identify aberrantly expressed exons in single samples.

Principal findings: As a proof-of-principle, we tested the PAC strategy on human cancer samples of which the complete coding sequence of eight cancer genes had been screened for mutations. PAC detected all seven exon-skipping mutants among 12 cancer cell lines. PAC also identified exon-skipping mutants in clinical cancer specimens although detection was compromised due to heterogeneous (wild-type) transcript expression. PAC reduced the number of candidate genes/exons for subsequent mutational analysis by two to three orders of magnitude and had a substantial true positive rate. Importantly, of 112 randomly selected outlier exons, sequence analysis identified two novel exon skipping events, two novel base changes and 21 previously reported base changes (SNPs).

Conclusions: The ability of PAC to enrich for mutated transcripts and to identify known and novel genetic changes confirms its suitability as a strategy to identify candidate cancer genes.

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PAC Identification of novel genetic changes.(A) PAC detection of novel genetic changes in EGFR. PAC predicted skipping of the last four exons of GBM157 and the 5′ end of exon 17 in GBM172. Semi-quantitative PCR on genomic DNA confirmed the deletion in GBM157 (not shown). (B) PAC predicts skipping of exon 30 in the FCGBP gene in GBM60. (C) RT-PCR confirmed the FCGBP exon skipping event in GBM60; other tumors did not show this exon skipping. (D) Direct sequencing identified a single base change in EGFR in GBM172 (as predicted by PAC, see Fig. 6A). (E) Confirmation of a PAC predicted change in the TLE2 gene in GBM60. The nucleotide substitution overlaps with individual probes of the probe set.
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pone-0003007-g006: PAC Identification of novel genetic changes.(A) PAC detection of novel genetic changes in EGFR. PAC predicted skipping of the last four exons of GBM157 and the 5′ end of exon 17 in GBM172. Semi-quantitative PCR on genomic DNA confirmed the deletion in GBM157 (not shown). (B) PAC predicts skipping of exon 30 in the FCGBP gene in GBM60. (C) RT-PCR confirmed the FCGBP exon skipping event in GBM60; other tumors did not show this exon skipping. (D) Direct sequencing identified a single base change in EGFR in GBM172 (as predicted by PAC, see Fig. 6A). (E) Confirmation of a PAC predicted change in the TLE2 gene in GBM60. The nucleotide substitution overlaps with individual probes of the probe set.

Mentions: One of the PAC identified novel exon skipping events was predicted to result in a deletion of the four 3′ end exons of EGFR (Fig. 6A). This exon-skipping event was due to a genomic deletion as determined using semi quantitative PCR on genomic tumor DNA. Compared to the 5′ end of the EGFR locus in GBM157, the 3′ end showed less amplification (ΔCt −2.5) whereas other samples showed equal amplification between the 5′ and 3′ end of the gene (ΔCt 0.3±1.9). Similar 3′ deletions in EGFR have been observed previously in gliomas [19]. The second exon-skipping event predicted by PAC would result in a deletion of exon 30 in the FCGBP cDNA (Fig. 6B). This deletion will cause a frameshift that is predicted to result in a truncated protein. The absence of exon 30 was confirmed by RT-PCR and sequence analysis (Fig. 6C). Novel identified single base changes include a single base change 1934C>G (S645C) in the EGFR gene, (Fig. 6A and D), and a single base change 946G>A (G316R) in the TLE2 gene (Fig. 6E). The G316R (946G>A) mutation in TLE2 is rendered “pathological” by PMut (mmb2.pcb.ub.es:8080/PMut/) and “not tolerated” by SIFT BLink (blocks.fhcrc.org/sift/SIFT_BLink_submit.html). In summary, the novel exon skipping events and base changes identified by analysis of a selected set of outlier exons confirms the suitability of PAC to identify candidate cancer genes.


Exon expression arrays as a tool to identify new cancer genes.

Schutte M, Elstrodt F, Bralten LB, Nagel JH, Duijm E, Hollestelle A, Vuerhard MJ, Wasielewski M, Peeters JK, van der Spek P, Sillevis Smitt PA, French PJ - PLoS ONE (2007)

PAC Identification of novel genetic changes.(A) PAC detection of novel genetic changes in EGFR. PAC predicted skipping of the last four exons of GBM157 and the 5′ end of exon 17 in GBM172. Semi-quantitative PCR on genomic DNA confirmed the deletion in GBM157 (not shown). (B) PAC predicts skipping of exon 30 in the FCGBP gene in GBM60. (C) RT-PCR confirmed the FCGBP exon skipping event in GBM60; other tumors did not show this exon skipping. (D) Direct sequencing identified a single base change in EGFR in GBM172 (as predicted by PAC, see Fig. 6A). (E) Confirmation of a PAC predicted change in the TLE2 gene in GBM60. The nucleotide substitution overlaps with individual probes of the probe set.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2500185&req=5

pone-0003007-g006: PAC Identification of novel genetic changes.(A) PAC detection of novel genetic changes in EGFR. PAC predicted skipping of the last four exons of GBM157 and the 5′ end of exon 17 in GBM172. Semi-quantitative PCR on genomic DNA confirmed the deletion in GBM157 (not shown). (B) PAC predicts skipping of exon 30 in the FCGBP gene in GBM60. (C) RT-PCR confirmed the FCGBP exon skipping event in GBM60; other tumors did not show this exon skipping. (D) Direct sequencing identified a single base change in EGFR in GBM172 (as predicted by PAC, see Fig. 6A). (E) Confirmation of a PAC predicted change in the TLE2 gene in GBM60. The nucleotide substitution overlaps with individual probes of the probe set.
Mentions: One of the PAC identified novel exon skipping events was predicted to result in a deletion of the four 3′ end exons of EGFR (Fig. 6A). This exon-skipping event was due to a genomic deletion as determined using semi quantitative PCR on genomic tumor DNA. Compared to the 5′ end of the EGFR locus in GBM157, the 3′ end showed less amplification (ΔCt −2.5) whereas other samples showed equal amplification between the 5′ and 3′ end of the gene (ΔCt 0.3±1.9). Similar 3′ deletions in EGFR have been observed previously in gliomas [19]. The second exon-skipping event predicted by PAC would result in a deletion of exon 30 in the FCGBP cDNA (Fig. 6B). This deletion will cause a frameshift that is predicted to result in a truncated protein. The absence of exon 30 was confirmed by RT-PCR and sequence analysis (Fig. 6C). Novel identified single base changes include a single base change 1934C>G (S645C) in the EGFR gene, (Fig. 6A and D), and a single base change 946G>A (G316R) in the TLE2 gene (Fig. 6E). The G316R (946G>A) mutation in TLE2 is rendered “pathological” by PMut (mmb2.pcb.ub.es:8080/PMut/) and “not tolerated” by SIFT BLink (blocks.fhcrc.org/sift/SIFT_BLink_submit.html). In summary, the novel exon skipping events and base changes identified by analysis of a selected set of outlier exons confirms the suitability of PAC to identify candidate cancer genes.

Bottom Line: An estimated 10-20% of cancer-related gene mutations result in skipping of one or more exons in the encoded transcripts.PAC reduced the number of candidate genes/exons for subsequent mutational analysis by two to three orders of magnitude and had a substantial true positive rate.Importantly, of 112 randomly selected outlier exons, sequence analysis identified two novel exon skipping events, two novel base changes and 21 previously reported base changes (SNPs).

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Josephine Nefkens Institute, Erasmus University Medical Center, Rotterdam, The Netherlands. a.schutte@erasmusmc.nl

ABSTRACT

Background: Identification of genes that are causally implicated in oncogenesis is a major goal in cancer research. An estimated 10-20% of cancer-related gene mutations result in skipping of one or more exons in the encoded transcripts. Here we report on a strategy to screen in a global fashion for such exon-skipping events using PAttern based Correlation (PAC). The PAC algorithm has been used previously to identify differentially expressed splice variants between two predefined subgroups. As genetic changes in cancer are sample specific, we tested the ability of PAC to identify aberrantly expressed exons in single samples.

Principal findings: As a proof-of-principle, we tested the PAC strategy on human cancer samples of which the complete coding sequence of eight cancer genes had been screened for mutations. PAC detected all seven exon-skipping mutants among 12 cancer cell lines. PAC also identified exon-skipping mutants in clinical cancer specimens although detection was compromised due to heterogeneous (wild-type) transcript expression. PAC reduced the number of candidate genes/exons for subsequent mutational analysis by two to three orders of magnitude and had a substantial true positive rate. Importantly, of 112 randomly selected outlier exons, sequence analysis identified two novel exon skipping events, two novel base changes and 21 previously reported base changes (SNPs).

Conclusions: The ability of PAC to enrich for mutated transcripts and to identify known and novel genetic changes confirms its suitability as a strategy to identify candidate cancer genes.

Show MeSH
Related in: MedlinePlus