Mutated tumor alleles are expressed according to their DNA frequency

Abstract

The transcription of tumor mutations from DNA into RNA has implications for biology, epigenetics and clinical practice. It is not clear if mutations are in general transcribed and, if so, at what proportion to the wild-type allele. Here, we examined the correlation between DNA mutation allele frequency and RNA mutation allele frequency. We sequenced the exome and transcriptome of tumor cell lines with large copy number variations, identified heterozygous single nucleotide mutations and absolute DNA copy number, and determined the corresponding DNA and RNA mutation allele fraction. We found that 99% of the DNA mutations in expressed genes are expressed as RNA. Moreover, we found a high correlation between the DNA and RNA mutation allele frequency. Exceptions are mutations that cause premature termination codons and therefore activate nonsense-mediated decay. Beyond this, we did not find evidence of any wide-scale mechanism, such as allele-specific epigenetic silencing, preferentially promoting mutated or wild-type alleles. In conclusion, our data strongly suggest that genes are equally transcribed from all alleles, mutated and wild-type, and thus transcribed in proportion to their DNA allele frequency.

Figure 1. DNA and RNA mutation allele frequency and copy number in the CT26 mouse colon cancer cell line.

(A and B): the sequence content and normalized read counts from the (A) BALB/cJ germline and (B) CT26 tumor DNA reads at the exemplary T > G Eif4g2 mutation locus (chr7, 118,222,833). Four nucleotides are shown on either side of the mutation. The exome of each sample (germline and tumor) was sequenced in duplicate. The normalized total number of reads overlapping the mutation locus is shown for each sample and replicate. The replicates show similar total counts and mutation frequencies. (C) The DNA gene copy number and DNA mutation allele frequency of the 3023 SNVs in CT26. Symbols and colors represent different chromosomes. Black crosses mark allowable mutation allele frequency and copy number combinations. The mutations with 100% allele frequency are homozygous mutations. (D) The sequence content and normalized read counts from the CT26 tumor RNA reads at the T > G Eif4g2 mutated locus. The CT26 transcriptome was sequenced in duplicate; the replicates show similar total counts and mutation frequencies. (E) The DNA and RNA mutation allele frequency for the 697 SNVs covered by at least 10 reads. SNVs are colored according to identified gene DNA copy number (CN), as determined in (C).

Figure 2. The DNA and RNA mutation allele frequency in CT26, B16F10 and 4T1 cells.

The marker color and size are determined by the total number of RNA reads (mutation containing plus wild-type containing reads) that overlap the SNV coordinate.

Figure 3. The impact of PTC-containing mutations on the CT26, B16F10 and 4T1 transcriptomes.

Red circles mark PTC-causing mutations in non-last-exons; green squares represent PTC-causing mutations in last exons.

Figure 4. Mutation frequency imbalance (RNA-DNA).

(A) A comparison of high and low expressed mutations based on mutations with at least 65 read coverage (blue, n = 179) or less than 15 read coverage (green, n = 180). The cutoffs were selected such that the sets have similar number of members. The means are similar but the variance in the low expression group is much higher. (B) The mutation frequency imbalance of non-synonymous (magenta, n = 596) and silent (red, n = 286) mutations. (C) The non-PTC (black, n = 955), PTC not-last-exon (red, n = 19) and PTC last-exon (green, n = 6) mutation sets and p-values.