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The application of nonsense-mediated mRNA decay inhibition to the identification of breast cancer susceptibility genes

BACKGROUND: Identification of novel, highly penetrant, breast cancer susceptibility genes will require the application of additional strategies beyond that of traditional linkage and candidate gene approaches. Approximately one-third of inherited genetic diseases, including breast cancer susceptibil...

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Autores principales: Johnson, Julie K, Waddell, Nic, Chenevix-Trench, Georgia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3409022/
https://www.ncbi.nlm.nih.gov/pubmed/22703186
http://dx.doi.org/10.1186/1471-2407-12-246
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author Johnson, Julie K
Waddell, Nic
Chenevix-Trench, Georgia
author_facet Johnson, Julie K
Waddell, Nic
Chenevix-Trench, Georgia
author_sort Johnson, Julie K
collection PubMed
description BACKGROUND: Identification of novel, highly penetrant, breast cancer susceptibility genes will require the application of additional strategies beyond that of traditional linkage and candidate gene approaches. Approximately one-third of inherited genetic diseases, including breast cancer susceptibility, are caused by frameshift or nonsense mutations that truncate the protein product [1]. Transcripts harbouring premature termination codons are selectively and rapidly degraded by the nonsense-mediated mRNA decay (NMD) pathway. Blocking the NMD pathway in any given cell will stabilise these mutant transcripts, which can then be detected using gene expression microarrays. This technique, known as gene identification by nonsense-mediated mRNA decay inhibition (GINI), has proved successful in identifying sporadic nonsense mutations involved in many different cancer types. However, the approach has not yet been applied to identify germline mutations involved in breast cancer. We therefore attempted to use GINI on lymphoblastoid cell lines (LCLs) from multiple-case, non- BRCA1/2 breast cancer families in order to identify additional high-risk breast cancer susceptibility genes. METHODS: We applied GINI to a total of 24 LCLs, established from breast-cancer affected and unaffected women from three multiple-case non-BRCA1/2 breast cancer families. We then used Illumina gene expression microarrays to identify transcripts stabilised by the NMD inhibition. RESULTS: The expression profiling identified a total of eight candidate genes from these three families. One gene, PPARGC1A, was a candidate in two separate families. We performed semi-quantitative real-time reverse transcriptase PCR of all candidate genes but only PPARGC1A showed successful validation by being stabilised in individuals with breast cancer but not in many unaffected members of the same family. Sanger sequencing of all coding and splice site regions of PPARGC1A did not reveal any protein truncating mutations. Haplotype analysis using short tandem repeat microsatellite markers did not indicate the presence of a haplotype around PPARGC1A which segregated with disease in the family. CONCLUSIONS: The application of the GINI method to LCLs to identify transcripts harbouring germline truncating mutations is challenging due to a number of factors related to cell type, microarray sensitivity and variations in NMD efficiency.
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spelling pubmed-34090222012-08-01 The application of nonsense-mediated mRNA decay inhibition to the identification of breast cancer susceptibility genes Johnson, Julie K Waddell, Nic Chenevix-Trench, Georgia BMC Cancer Research Article BACKGROUND: Identification of novel, highly penetrant, breast cancer susceptibility genes will require the application of additional strategies beyond that of traditional linkage and candidate gene approaches. Approximately one-third of inherited genetic diseases, including breast cancer susceptibility, are caused by frameshift or nonsense mutations that truncate the protein product [1]. Transcripts harbouring premature termination codons are selectively and rapidly degraded by the nonsense-mediated mRNA decay (NMD) pathway. Blocking the NMD pathway in any given cell will stabilise these mutant transcripts, which can then be detected using gene expression microarrays. This technique, known as gene identification by nonsense-mediated mRNA decay inhibition (GINI), has proved successful in identifying sporadic nonsense mutations involved in many different cancer types. However, the approach has not yet been applied to identify germline mutations involved in breast cancer. We therefore attempted to use GINI on lymphoblastoid cell lines (LCLs) from multiple-case, non- BRCA1/2 breast cancer families in order to identify additional high-risk breast cancer susceptibility genes. METHODS: We applied GINI to a total of 24 LCLs, established from breast-cancer affected and unaffected women from three multiple-case non-BRCA1/2 breast cancer families. We then used Illumina gene expression microarrays to identify transcripts stabilised by the NMD inhibition. RESULTS: The expression profiling identified a total of eight candidate genes from these three families. One gene, PPARGC1A, was a candidate in two separate families. We performed semi-quantitative real-time reverse transcriptase PCR of all candidate genes but only PPARGC1A showed successful validation by being stabilised in individuals with breast cancer but not in many unaffected members of the same family. Sanger sequencing of all coding and splice site regions of PPARGC1A did not reveal any protein truncating mutations. Haplotype analysis using short tandem repeat microsatellite markers did not indicate the presence of a haplotype around PPARGC1A which segregated with disease in the family. CONCLUSIONS: The application of the GINI method to LCLs to identify transcripts harbouring germline truncating mutations is challenging due to a number of factors related to cell type, microarray sensitivity and variations in NMD efficiency. BioMed Central 2012-06-15 /pmc/articles/PMC3409022/ /pubmed/22703186 http://dx.doi.org/10.1186/1471-2407-12-246 Text en Copyright ©2012 Johnson et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Johnson, Julie K
Waddell, Nic
Chenevix-Trench, Georgia
The application of nonsense-mediated mRNA decay inhibition to the identification of breast cancer susceptibility genes
title The application of nonsense-mediated mRNA decay inhibition to the identification of breast cancer susceptibility genes
title_full The application of nonsense-mediated mRNA decay inhibition to the identification of breast cancer susceptibility genes
title_fullStr The application of nonsense-mediated mRNA decay inhibition to the identification of breast cancer susceptibility genes
title_full_unstemmed The application of nonsense-mediated mRNA decay inhibition to the identification of breast cancer susceptibility genes
title_short The application of nonsense-mediated mRNA decay inhibition to the identification of breast cancer susceptibility genes
title_sort application of nonsense-mediated mrna decay inhibition to the identification of breast cancer susceptibility genes
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3409022/
https://www.ncbi.nlm.nih.gov/pubmed/22703186
http://dx.doi.org/10.1186/1471-2407-12-246
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