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Massive parallel DNA pyrosequencing analysis of the tumor suppressor BRG1/SMARCA4 in lung primary tumors
Salvador Rodriguez-Nieto1, Andres Cañada, Eva Pros
1Genes and Cancer Group, Cancer Epigenetics and Biology Program-PEBC (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain.
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.
Insights
Ultra-deep sequencing revealed SMARCA4 mutations in lung tumors, previously missed by standard methods. This tumor suppressor gene is frequently inactivated in lung cancer, impacting disease development.
Area of Science:
- Oncology
- Genetics
- Molecular Biology
Background:
- The tumor suppressor gene SMARCA4 (BRG1) is crucial for chromatin remodeling.
- SMARCA4 alterations are frequently observed in lung cancer cell lines but less so in primary tumors.
- Ultra-deep sequencing offers enhanced sensitivity for detecting genetic variations.
Purpose of the Study:
- To investigate SMARCA4 mutations in lung primary tumors using ultra-deep pyrosequencing.
- To compare the sensitivity of ultra-deep pyrosequencing with standard sequencing methods for SMARCA4 mutation detection.
- To assess the frequency and significance of SMARCA4 biallelic inactivation in lung cancer development.
Main Methods:
- Screening of the entire SMARCA4 coding region in 12 lung tumors using ultra-deep pyrosequencing.
- Comparison of mutation detection rates between standard automatic-fluorescence-based sequencing and pyrosequencing.
- Confirmation of splice site variants using individual cloning of PCR products.
Main Results:
- Ultra-deep pyrosequencing identified additional SMARCA4 variants compared to standard sequencing.
- One somatic splice site mutation was confirmed, present at a low frequency (10%) suggesting tumor heterogeneity or normal cell contamination.
- The study determined the sensitivity and limitations of ultra-deep pyrosequencing technology.
Conclusions:
- SMARCA4 is biallelically inactivated in a significant proportion of lung primary tumors, not just cell lines.
- SMARCA4 is a critical gene in the pathogenesis of lung cancer.
- Ultra-deep sequencing enhances the detection of low-frequency mutations in cancer research.
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