Increased frequency of single base substitutions in a population of transcripts expressed in cancer cells

Laurent Bianchetti,Olivier Poch,David Kieffer,Rémi Féderkeil

doi:10.1186/1471-2407-12-509

Abstract

BackgroundSingle Base Substitutions (SBS) that alter transcripts expressed in cancer originate from somatic mutations. However, recent studies report SBS in transcripts that are not supported by the genomic DNA of tumor cells.MethodsWe used sequence based whole genome expression profiling, namely Long-SAGE (L-SAGE) and Tag-seq (a combination of L-SAGE and deep sequencing), and computational methods to identify transcripts with greater SBS frequencies in cancer. Millions of tags produced by 40 healthy and 47 cancer L-SAGE experiments were compared to 1,959 Reference Tags (RT), i.e. tags matching the human genome exactly once. Similarly, tens of millions of tags produced by 7 healthy and 8 cancer Tag-seq experiments were compared to 8,572 RT. For each transcript, SBS frequencies in healthy and cancer cells were statistically tested for equality.ResultsIn the L-SAGE and Tag-seq experiments, 372 and 4,289 transcripts respectively, showed greater SBS frequencies in cancer. Increased SBS frequencies could not be attributed to known Single Nucleotide Polymorphisms (SNP), catalogued somatic mutations or RNA-editing enzymes. Hypothesizing that Single Tags (ST), i.e. tags sequenced only once, were indicators of SBS, we observed that ST proportions were heterogeneously distributed across Embryonic Stem Cells (ESC), healthy differentiated and cancer cells. ESC had the lowest ST proportions, whereas cancer cells had the greatest. Finally, in a series of experiments carried out on a single patient at 1 healthy and 3 consecutive tumor stages, we could show that SBS frequencies increased during cancer progression.ConclusionIf the mechanisms generating the base substitutions could be known, increased SBS frequency in transcripts would be a new useful biomarker of cancer. With the reduction of sequencing cost, sequence based whole genome expression profiling could be used to characterize increased SBS frequency in patient’s tumor and aid diagnostic.

Highlights

Single Base Substitutions (SBS) that alter transcripts expressed in cancer originate from somatic mutations
Groups of healthy and cancer experiments 87 L-SAGE and 15 Tag-seq experiments were selected on the NCBI Gene Expression Omnibus (GEO) repository [21]
Since the total number of tags produced by L-SAGE and Tag-seq experiments were dramatically different and because the sequencing error rates of Sanger and deep sequencing methods may be unequal, L-SAGE and Tag-seq tags were processed using the same bioinformatics workflow but separately (Figure 1)

Summary

Introduction

Single Base Substitutions (SBS) that alter transcripts expressed in cancer originate from somatic mutations. Since most EST are 3’ fragments of mRNA sequences, increased SBS in cancer was detected at the 3’ boundary of mRNA These SBS could not be explained by known SNP and were unlikely the result of somatic mutations or RNA-editing enzymes. The concept of transcriptional infidelity (TI) was proposed: i) TI introduces non-random base variations in RNA sequences that are not supported by the genome ii) TI exists in both healthy and cancer cells, but is greater in cancer. The genomic sequences of the tumor and the healthy cells were not simultaneously available in our study, these SBS could not be attributed to known SNP, catalogued cancer related somatic mutations, and known APOBEC1 or ADAR editing. Focusing on a series of 4 L-SAGE experiments carried out on the biopsies of a single patient at 1 healthy and 3 consecutive tumor stages, we were able to demonstrate that SBS frequencies significantly increased during cancer progression

Methods

Results

Discussion

Conclusion