Team suggests disease susceptibility studies should focus on mRNA sequence variations as well as DNA polymorphisms.

The finding that one stretch of coding DNA doesn’t necessarily get transcribed into a matching stretch of RNA has led researchers to query whether RNA sequence variation could impact disease susceptibility or manifestation.

Studying factors related to disease susceptibility at the genetic level has to date largely focused on looking at variations in the genes themselves or measuring levels of corresponding protein production. Studies by Vivan G. Cheung, Ph.D., at the University of Pennsylvania School of Medicine in Philadelphia, and colleagues, suggest, however, that RNA sequence variations may also be relevant.

When the researchers compared the RNA sequences in human B cells with the corresponding DNA sequences in the same cells, they found 28,766 DNA-RNA mismatch events at over 10,000 exonic sites in 4,741 genes. Each specific mismatch was observed in the B cells of at least two individuals from the original cohort of 27 people and was subsequently identified in primary skin cells and brain tissues from a separate set of individuals.

All 12 possible categories of discordances were observed, and in a number of cases peptides were generated from the discordant RNAs that didn’t correspond to the DNAs. The researchers report their results in Science Express in a paper titled “Widespread RNA and DNA Sequence Differences in the Human Transcriptome.”

It is widely assumed that a sequence of an mRNA exactly reflects that of the DNA from which it was transcribed, and this assumed precision is important given that mRNA serves as the template for protein synthesis, Dr. Cheung and colleagues explain. Although errors in transcription can occur they are relatively rare, and RNA-DNA differences that are known to occur as a result of RNA editing are effected by enzymes that target the mRNA post-trancriptionally.

As a result of the expected one-to-one relationship between DNA and its mRNA transcript, studies into the genetics of disease susceptibility have focused largely on DNA sequence polymorphisms, while the analysis of mRNA and proteins has centered largely on levels of their expression, rather than on any potential RNA sequence differences among individuals.

The researchers have now poured significant doubt on this basic tenet of DNA-to-RNA sequence equivalence. They compared the DNA and mRNA sequences from B cells in 27 unrelated individuals who are part of the International HapMap Consortium and 1000 Genomes Projects.  Of the 28,766 events identified, nearly 7,000 were A-G events, which could be the result of deamination by ADAR, and another 1,220 were C-T differences which could also be mediated by a deaminase, the researchers admit.

However, 43% if the differences (12,507) were transversions at 10,210 exonic sites, which couldn’t have resulted from classic deaminase-mediated editing. The mechanisms behind these sequence differences, which they termed RNA-DNA differences (RDDs) remains unknown.

Significantly, the researchers add, “these sites where RNA sequences differ from the corresponding DNA sequences appear to be non-random since the identical differences were found in multiple individuals.” In fact, 80% of the RDD sites were found in at least 50% of the test individuals. Also of importance was the mass spectrometry-based confirmation that sequences corresponding to at least some of the RNAs were translated into peptides.

Interestingly, the mismatched DNA and RNA sequences were not spread evenly across the 10,210 exonic sites. Chromosome 19 had the most and chromosome 13 the fewest. The average number of exonic DNA-RNA mismatches per person among the Gencode genes was just over a thousand but in some people it was in the low-100s and in others nearly 2,000.

The team validated its findings experimentally by Sanger sequencing of both DNA and RNA at 12 randomly selected sites in the B cells, primary skin, and brain cortex from multiple individuals as well as in tumor cells. B cells from the subjects were also regrown and DNA and mRNA extracted from the same aliquots of cells.

The consistent pattern of the observations suggests that the RDDs have biological significance and are not just noise, the authors conclude.  At nearly all RDD sites, we observed only one RDD type across cell types and in different individuals.

” At present, however, the underlying mechanisms for these events remain largely unknown,” the investigators admit. “For most of the cases we do not know yet whether a different base was incorporated into the RNA during transcription or if these events occur post-transcriptionally.”

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