title: Massive parallel DNA pyrosequencing analysis of the tumor suppressor BRG1/SMARCA4 in lung primary tumors.
abstract: The tumor suppressor gene, SMARCA4 (or BRG1), which encodes the ATPase component of the chromatin remodeling complex SWI/SNF, is commonly inactivated by mutations and deletions in lung cancer cell lines. However, SMARCA4 alterations appear to be rare in lung primary tumors. Ultra-deep sequencing technologies provide a promising alternative to achieve a sensitivity superior to that of current sequencing strategies. Here we used ultra-deep pyrosequencing to screen for mutations over the entire SMARCA4 coding region in 12 lung tumors without detectable BRG1 protein. While automatic-fluorescence-based sequencing detected one somatic mutation (p.K586X), the pyrosequencing revealed additional variants, thus increasing the sensitivity. One of the variants, which affected a consensus splice site, was confirmed by individual cloning of PCR products, ruling out the possibility of PCR or pyrosequencing artifacts. This mutation, confirmed to be somatic, was present at a frequency of ten percent, suggesting normal cell contamination in the tumor. Our analysis also allowed us to determine the sensitivity and to identify some limitations of the technology. In conclusion, in addition to cell lines, SMARCA4 is biallelically inactivated in a significant proportion of lung primary tumors, thereby constituting one of the most important genes contributing to the development of this type of cancer.
authors.affiliation: Genes and Cancer Group, Cancer Epigenetics and Biology Program-PEBC (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain.
authors.forename: Salvador
authors.initials: S
authors.lastname: Rodriguez-Nieto
authors.forename: Andres
authors.initials: A
authors.lastname: Cañada
authors.forename: Eva
authors.initials: E
authors.lastname: Pros
authors.forename: Ana I
authors.initials: AI
authors.lastname: Pinto
authors.forename: Juan
authors.initials: J
authors.lastname: Torres-Lanzas
authors.forename: Fernando
authors.initials: F
authors.lastname: Lopez-Rios
authors.forename: Lydia
authors.initials: L
authors.lastname: Sanchez-Verde
authors.forename: David G
authors.initials: DG
authors.lastname: Pisano
authors.forename: Montse
authors.initials: M
authors.lastname: Sanchez-Cespedes
mesh_terms: D045744:Cell Line, Tumor; D004265:DNA Helicases / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008175:Lung Neoplasms / Q000175:diagnosis* / Q000235:genetics; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*; D014157:Transcription Factors / Q000235:genetics*
chemical_list: D009687:Nuclear Proteins; D014157:Transcription Factors; C084108:SMARCA4 protein, human; D004265:DNA Helicases