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Targeted cancer exome sequencing reveals recurrent mutations in myeloproliferative neoplasms.

Tenedini E, Bernardis I, Artusi V, Artuso L, Roncaglia E, Guglielmelli P, Pieri L, Bogani C, Biamonte F, Rotunno G, Mannarelli C, Bianchi E, Pancrazzi A, Fanelli T, Malagoli Tagliazucchi G, Ferrari S, Manfredini R, Vannucchi AM, Tagliafico E, AGIMM investigato - Leukemia (2013)

Bottom Line: Excluding the genes already described in MPN, for 8 genes (SCRIB, MIR662, BARD1, TCF12, FAT4, DAP3, POLG and NRAS), we demonstrated a mutation frequency between 3 and 8%.We also found that mutations at codon 12 of NRAS (NRASG12V and NRASG12D) were significantly associated, for primary myelofibrosis (PMF), with highest dynamic international prognostic scoring system (DIPSS)-plus score categories.This association was then confirmed in 66 additional PMF patients composing a final dataset of 168 PMF showing a NRAS mutation frequency of 4.7%, which was associated with a worse outcome, as defined by the DIPSS plus score.

View Article: PubMed Central - PubMed

Affiliation: 1] Center for Genome Research, University of Modena and Reggio Emilia, Modena, Italy [2] Life Sciences Department, University of Modena and Reggio Emilia, Modena, Italy [3] Center for Regenerative Medicine 'Stefano Ferrari', University of Modena and Reggio Emilia, Modena, Italy.

ABSTRACT
With the intent of dissecting the molecular complexity of Philadelphia-negative myeloproliferative neoplasms (MPN), we designed a target enrichment panel to explore, using next-generation sequencing (NGS), the mutational status of an extensive list of 2000 cancer-associated genes and microRNAs. The genomic DNA of granulocytes and in vitro-expanded CD3+T-lymphocytes, as a germline control, was target-enriched and sequenced in a learning cohort of 20 MPN patients using Roche 454 technology. We identified 141 genuine somatic mutations, most of which were not previously described. To test the frequency of the identified variants, a larger validation cohort of 189 MPN patients was additionally screened for these mutations using Ion Torrent AmpliSeq NGS. Excluding the genes already described in MPN, for 8 genes (SCRIB, MIR662, BARD1, TCF12, FAT4, DAP3, POLG and NRAS), we demonstrated a mutation frequency between 3 and 8%. We also found that mutations at codon 12 of NRAS (NRASG12V and NRASG12D) were significantly associated, for primary myelofibrosis (PMF), with highest dynamic international prognostic scoring system (DIPSS)-plus score categories. This association was then confirmed in 66 additional PMF patients composing a final dataset of 168 PMF showing a NRAS mutation frequency of 4.7%, which was associated with a worse outcome, as defined by the DIPSS plus score.

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Circular diagram of mutations found in MPN. Chromosomes are illustrated in the outer perimeter. Grey dots show the ‘cancer exome' regions of the NimbleGen panel, whereas the histograms show the captured (blue) and failed (red) target regions. MicroRNA or Gene Symbol with amino acidic change refers to the variants found in our cohort.
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fig2: Circular diagram of mutations found in MPN. Chromosomes are illustrated in the outer perimeter. Grey dots show the ‘cancer exome' regions of the NimbleGen panel, whereas the histograms show the captured (blue) and failed (red) target regions. MicroRNA or Gene Symbol with amino acidic change refers to the variants found in our cohort.

Mentions: Using this strategy, we estimated a very low sequencing error rate (<1%), and we finally confirmed 136 genuine somatic, non-synonymous mutations affecting 121 genes. Twenty-five percent of these mutations are indexed in the dbSNP archive, and 2% of these specific variants are listed in COSMIC catalogue. The majority of mutations (89%) were estimated to be ‘damaging' by at least 1 of the 5 algorithms that we used to investigate disease-causing potential (PolyPhen2, SIFT, Provean, Mutation Taster, LTR) (Supplementary Table 4 and Figure 2).


Targeted cancer exome sequencing reveals recurrent mutations in myeloproliferative neoplasms.

Tenedini E, Bernardis I, Artusi V, Artuso L, Roncaglia E, Guglielmelli P, Pieri L, Bogani C, Biamonte F, Rotunno G, Mannarelli C, Bianchi E, Pancrazzi A, Fanelli T, Malagoli Tagliazucchi G, Ferrari S, Manfredini R, Vannucchi AM, Tagliafico E, AGIMM investigato - Leukemia (2013)

Circular diagram of mutations found in MPN. Chromosomes are illustrated in the outer perimeter. Grey dots show the ‘cancer exome' regions of the NimbleGen panel, whereas the histograms show the captured (blue) and failed (red) target regions. MicroRNA or Gene Symbol with amino acidic change refers to the variants found in our cohort.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4017260&req=5

fig2: Circular diagram of mutations found in MPN. Chromosomes are illustrated in the outer perimeter. Grey dots show the ‘cancer exome' regions of the NimbleGen panel, whereas the histograms show the captured (blue) and failed (red) target regions. MicroRNA or Gene Symbol with amino acidic change refers to the variants found in our cohort.
Mentions: Using this strategy, we estimated a very low sequencing error rate (<1%), and we finally confirmed 136 genuine somatic, non-synonymous mutations affecting 121 genes. Twenty-five percent of these mutations are indexed in the dbSNP archive, and 2% of these specific variants are listed in COSMIC catalogue. The majority of mutations (89%) were estimated to be ‘damaging' by at least 1 of the 5 algorithms that we used to investigate disease-causing potential (PolyPhen2, SIFT, Provean, Mutation Taster, LTR) (Supplementary Table 4 and Figure 2).

Bottom Line: Excluding the genes already described in MPN, for 8 genes (SCRIB, MIR662, BARD1, TCF12, FAT4, DAP3, POLG and NRAS), we demonstrated a mutation frequency between 3 and 8%.We also found that mutations at codon 12 of NRAS (NRASG12V and NRASG12D) were significantly associated, for primary myelofibrosis (PMF), with highest dynamic international prognostic scoring system (DIPSS)-plus score categories.This association was then confirmed in 66 additional PMF patients composing a final dataset of 168 PMF showing a NRAS mutation frequency of 4.7%, which was associated with a worse outcome, as defined by the DIPSS plus score.

View Article: PubMed Central - PubMed

Affiliation: 1] Center for Genome Research, University of Modena and Reggio Emilia, Modena, Italy [2] Life Sciences Department, University of Modena and Reggio Emilia, Modena, Italy [3] Center for Regenerative Medicine 'Stefano Ferrari', University of Modena and Reggio Emilia, Modena, Italy.

ABSTRACT
With the intent of dissecting the molecular complexity of Philadelphia-negative myeloproliferative neoplasms (MPN), we designed a target enrichment panel to explore, using next-generation sequencing (NGS), the mutational status of an extensive list of 2000 cancer-associated genes and microRNAs. The genomic DNA of granulocytes and in vitro-expanded CD3+T-lymphocytes, as a germline control, was target-enriched and sequenced in a learning cohort of 20 MPN patients using Roche 454 technology. We identified 141 genuine somatic mutations, most of which were not previously described. To test the frequency of the identified variants, a larger validation cohort of 189 MPN patients was additionally screened for these mutations using Ion Torrent AmpliSeq NGS. Excluding the genes already described in MPN, for 8 genes (SCRIB, MIR662, BARD1, TCF12, FAT4, DAP3, POLG and NRAS), we demonstrated a mutation frequency between 3 and 8%. We also found that mutations at codon 12 of NRAS (NRASG12V and NRASG12D) were significantly associated, for primary myelofibrosis (PMF), with highest dynamic international prognostic scoring system (DIPSS)-plus score categories. This association was then confirmed in 66 additional PMF patients composing a final dataset of 168 PMF showing a NRAS mutation frequency of 4.7%, which was associated with a worse outcome, as defined by the DIPSS plus score.

Show MeSH
Related in: MedlinePlus