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Somatic mutation profiles of MSI and MSS colorectal cancer identified by whole exome next generation sequencing and bioinformatics analysis.

Timmermann B, Kerick M, Roehr C, Fischer A, Isau M, Boerno ST, Wunderlich A, Barmeyer C, Seemann P, Koenig J, Lappe M, Kuss AW, Garshasbi M, Bertram L, Trappe K, Werber M, Herrmann BG, Zatloukal K, Lehrach H, Schweiger MR - PLoS ONE (2010)

Bottom Line: Here we present the first work on whole exome NGS of primary colon cancers.Our bioinformatics filtering approach narrowed down the rate of most significant mutations to 359 for MSI and 45 for MSS CRCs with predicted altered protein functions.In both CRCs, MSI and MSS, we found somatic mutations in the intracellular kinase domain of bone morphogenetic protein receptor 1A, BMPR1A, a gene where so far germline mutations are associated with juvenile polyposis syndrome, and show that the mutations functionally impair the protein function.

View Article: PubMed Central - PubMed

Affiliation: Next Generation Sequencing Group, Max Planck Institute for Molecular Genetics, Berlin, Germany.

ABSTRACT

Background: Colorectal cancer (CRC) is with approximately 1 million cases the third most common cancer worldwide. Extensive research is ongoing to decipher the underlying genetic patterns with the hope to improve early cancer diagnosis and treatment. In this direction, the recent progress in next generation sequencing technologies has revolutionized the field of cancer genomics. However, one caveat of these studies remains the large amount of genetic variations identified and their interpretation.

Methodology/principal findings: Here we present the first work on whole exome NGS of primary colon cancers. We performed 454 whole exome pyrosequencing of tumor as well as adjacent not affected normal colonic tissue from microsatellite stable (MSS) and microsatellite instable (MSI) colon cancer patients and identified more than 50,000 small nucleotide variations for each tissue. According to predictions based on MSS and MSI pathomechanisms we identified eight times more somatic non-synonymous variations in MSI cancers than in MSS and we were able to reproduce the result in four additional CRCs. Our bioinformatics filtering approach narrowed down the rate of most significant mutations to 359 for MSI and 45 for MSS CRCs with predicted altered protein functions. In both CRCs, MSI and MSS, we found somatic mutations in the intracellular kinase domain of bone morphogenetic protein receptor 1A, BMPR1A, a gene where so far germline mutations are associated with juvenile polyposis syndrome, and show that the mutations functionally impair the protein function.

Conclusions/significance: We conclude that with deep sequencing of tumor exomes one may be able to predict the microsatellite status of CRC and in addition identify potentially clinically relevant mutations.

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Qualities of the targeted whole exome sequencing approach.(A) Venn diagram of captured exons of normal and tumor samples. Captured exons with at least one read were counted. (B) Representative normalized coverage-distribution plot. The fraction of bait-covered exons in the genome achieving coverages equal or lower than the normalized coverage is indicated on the x-axis. The mean coverage per exon was divided by the mean coverage of all exons.
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pone-0015661-g001: Qualities of the targeted whole exome sequencing approach.(A) Venn diagram of captured exons of normal and tumor samples. Captured exons with at least one read were counted. (B) Representative normalized coverage-distribution plot. The fraction of bait-covered exons in the genome achieving coverages equal or lower than the normalized coverage is indicated on the x-axis. The mean coverage per exon was divided by the mean coverage of all exons.

Mentions: We analyzed the complete exomes of more than 135,000 exons with single-read shotgun 454 sequencing (Figure 1, Figure S1, Figure S2, Table 2). To assess the effect of coverage depth on the sensitivity and specificity of sequence variant detection, genotype calls of the Affymetrix SNP array 6.0 were compared step-wise to the called nucleic acid positions and resulted in an accuracy of more than 99% (Figure S3). In addition to the SNP array, we used Sanger sequencing to confirm 23 selected mutations (Table S5, Figure S5).


Somatic mutation profiles of MSI and MSS colorectal cancer identified by whole exome next generation sequencing and bioinformatics analysis.

Timmermann B, Kerick M, Roehr C, Fischer A, Isau M, Boerno ST, Wunderlich A, Barmeyer C, Seemann P, Koenig J, Lappe M, Kuss AW, Garshasbi M, Bertram L, Trappe K, Werber M, Herrmann BG, Zatloukal K, Lehrach H, Schweiger MR - PLoS ONE (2010)

Qualities of the targeted whole exome sequencing approach.(A) Venn diagram of captured exons of normal and tumor samples. Captured exons with at least one read were counted. (B) Representative normalized coverage-distribution plot. The fraction of bait-covered exons in the genome achieving coverages equal or lower than the normalized coverage is indicated on the x-axis. The mean coverage per exon was divided by the mean coverage of all exons.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3008745&req=5

pone-0015661-g001: Qualities of the targeted whole exome sequencing approach.(A) Venn diagram of captured exons of normal and tumor samples. Captured exons with at least one read were counted. (B) Representative normalized coverage-distribution plot. The fraction of bait-covered exons in the genome achieving coverages equal or lower than the normalized coverage is indicated on the x-axis. The mean coverage per exon was divided by the mean coverage of all exons.
Mentions: We analyzed the complete exomes of more than 135,000 exons with single-read shotgun 454 sequencing (Figure 1, Figure S1, Figure S2, Table 2). To assess the effect of coverage depth on the sensitivity and specificity of sequence variant detection, genotype calls of the Affymetrix SNP array 6.0 were compared step-wise to the called nucleic acid positions and resulted in an accuracy of more than 99% (Figure S3). In addition to the SNP array, we used Sanger sequencing to confirm 23 selected mutations (Table S5, Figure S5).

Bottom Line: Here we present the first work on whole exome NGS of primary colon cancers.Our bioinformatics filtering approach narrowed down the rate of most significant mutations to 359 for MSI and 45 for MSS CRCs with predicted altered protein functions.In both CRCs, MSI and MSS, we found somatic mutations in the intracellular kinase domain of bone morphogenetic protein receptor 1A, BMPR1A, a gene where so far germline mutations are associated with juvenile polyposis syndrome, and show that the mutations functionally impair the protein function.

View Article: PubMed Central - PubMed

Affiliation: Next Generation Sequencing Group, Max Planck Institute for Molecular Genetics, Berlin, Germany.

ABSTRACT

Background: Colorectal cancer (CRC) is with approximately 1 million cases the third most common cancer worldwide. Extensive research is ongoing to decipher the underlying genetic patterns with the hope to improve early cancer diagnosis and treatment. In this direction, the recent progress in next generation sequencing technologies has revolutionized the field of cancer genomics. However, one caveat of these studies remains the large amount of genetic variations identified and their interpretation.

Methodology/principal findings: Here we present the first work on whole exome NGS of primary colon cancers. We performed 454 whole exome pyrosequencing of tumor as well as adjacent not affected normal colonic tissue from microsatellite stable (MSS) and microsatellite instable (MSI) colon cancer patients and identified more than 50,000 small nucleotide variations for each tissue. According to predictions based on MSS and MSI pathomechanisms we identified eight times more somatic non-synonymous variations in MSI cancers than in MSS and we were able to reproduce the result in four additional CRCs. Our bioinformatics filtering approach narrowed down the rate of most significant mutations to 359 for MSI and 45 for MSS CRCs with predicted altered protein functions. In both CRCs, MSI and MSS, we found somatic mutations in the intracellular kinase domain of bone morphogenetic protein receptor 1A, BMPR1A, a gene where so far germline mutations are associated with juvenile polyposis syndrome, and show that the mutations functionally impair the protein function.

Conclusions/significance: We conclude that with deep sequencing of tumor exomes one may be able to predict the microsatellite status of CRC and in addition identify potentially clinically relevant mutations.

Show MeSH
Related in: MedlinePlus