Transcriptome sequencing of malignant pleural mesothelioma tumors

*International Mesothelioma Program,
^†Division of Thoracic Surgery,
^§Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, 75 Francis Street, Boston, MA 02115;
^¶454 Life Sciences, Inc., 20 Commercial Street, Branford, CT 06405;
^‖National Center for Genome Resources (NCGR), 2935 Rodeo Park Drive East, Santa Fe, NM 87505;
**Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA 24060; and
^††RxGen, Inc., 100 Deepwood Drive, Hamden, CT 06517

Communicated by Elliott D. Kieff, Harvard University, Boston, MA, December 31, 2007 (received for review July 6, 2007)

Abstract

Cancers arise by the gradual accumulation of mutations in multiple genes. We now use shotgun pyrosequencing to characterize RNA mutations and expression levels unique to malignant pleural mesotheliomas (MPMs) and not present in control tissues. On average, 266 Mb of cDNA were sequenced from each of four MPMs, from a control pulmonary adenocarcinoma (ADCA), and from normal lung tissue. Previously observed differences in MPM RNA expression levels were confirmed. Point mutations were identified by using criteria that require the presence of the mutation in at least four reads and in both cDNA strands and the absence of the mutation from sequence databases, normal adjacent tissues, and other controls. In the four MPMs, 15 nonsynonymous mutations were discovered: 7 were point mutations, 3 were deletions, 4 were exclusively expressed as a consequence of imputed epigenetic silencing, and 1 was putatively expressed as a consequence of RNA editing. Notably, each MPM had a different mutation profile, and no mutated gene was previously implicated in MPM. Of the seven point mutations, three were observed in at least one tumor from 49 other MPM patients. The mutations were in genes that could be causally related to cancer and included XRCC6, PDZK1IP1, ACTR1A, and AVEN.

Footnotes

^‡To whom correspondence should be addressed. E-mail: dsugarbaker{at}partners.org
Author contributions: D.J.S., W.G.R., G.J.G., S.R.G., and R.B. designed research; G.J.G., L. Dong, A.D.R., G.M., J.N.G., L.R.C., M.-L.H., B.E.T., L. Du, and P.B. performed research; L. Dong, B.E.T., L. Du, S.F.K., N.A.M., A.D.F., and R.V.J. contributed new reagents/analytic tools; D.J.S., W.G.R., G.J.G., L. Dong, A.D.R., G.M., J.N.G., L.R.C., L. Du, S.F.K., N.A.M., A.D.F., R.V.J., S.R.G., and R.B. analyzed data; and D.J.S., W.G.R., G.J.G., S.F.K., R.V.J., S.R.G., and R.B. wrote the paper.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0712399105/DC1.
↵ ‡‡ SNVs are defined as single base substitutions that differ from the human mRNA reference sequence obtained from the NCBI RefSeq mRNA database. SNPs refer to those SNVs either present in the NCBI dbSNP database or observed in the patient's nontumor DNA or cDNA from blood.
Freely available online through the PNAS open access option.