Nonsense Mediated Decay (NMD) in Cancer Variant Interpretation
When cancer DNA sequencing is performed, one of the critical steps in processing the results is interpretation of the mutations that were detected. Some mutations are benign, while others are pathogenic (cancer-causing). The pathogenic mutations are the important ones to focus on, as they may have actionable implications for the patient's cancer treatment, such as targeted therapies, prognosis, or diagnosis. There are many pieces of information about a mutation that may give us clues to its pathogenicity. One of those clues is how the mutation affects the function of a protein. If the mutation in a gene that is a tumor-suppressor gene, meaning that it’s job is to prevent growth of cancer, then a mutation whose effect on the protein is “loss of function” is likely to cause cancer. The protein can’t do its job of preventing cancer growth, and as a result, cancer may grow. Understanding Nonsense Mediated Decay is a critical piece of determining if a mutation will have a “loss of function” effect on the protein.
What is NMD?
Nonsense Mediated Decay (NMD) is a form of mRNA surveillance, which is the human body’s way of finding and targeting defective mRNA transcripts for destruction before they produce proteins. NMD is the mRNA surveillance mechanism that targets transcripts containing a premature termination codon (PTC), and destroys them to prevent truncated proteins from being created. In other words, NMD guarantees that the mutation leads to a “loss of function,” because the protein won’t be created at all!
How does NMD Work?
When the intronic regions are spliced out of the RNA and exons join together, protein complexes called Exon Junction Complexes (EJCs) remain at the exon-exon boundaries.
During translation, when the ribosome walks along the RNA, it knocks off the EJCs as it encounters them. All EJCs past the 5’ UTR are removed. The Ribosome continues until it reaches a stop codon, and the stop codon recruits release factors that release the protein from the ribosome, and then the ribosome releases from the RNA.
In nonsense or frameshift mutations, a PTC is introduced. The ribosome won’t travel past the PTC and thus there may be additional EJCs beyond the PTC that never get knocked off by the ribosome.
The EJC recruits factors that identify and bind to the stalled ribosome, which mark the RNA molecule for destruction. So the RNA molecule is destroyed before it can produce any truncated proteins. This process is Nonsense Mediated Decay.
The Blind Spots of NMD (NMD Escape)
The NMD mechanism is not perfect. There are 4 known ways that a transcript with a PTC may survive despite NMD1,2,3. We call this concept NMD Escape:
The PTC is in the last exon. There are no remaining EJCs past the PTC, so there is no way to identify this as a faulty transcript.
The PTC is close to the last EJC (within ~50 bp). There is still an exon-exon junction after the PTC. However, the effective combined radius of the EJC and ribosome is ~50 bp, so that when the ribosome gets within that range of the EJC, the EJC gets knocked off even though the ribosome didn't translate all the way to the last junction. Similar to 1, since the last EJC gets knocked off, there are no EJC’s left.
The PTC is in a very long exon (>407 bp). Even though there are still EJCs remaining on the RNA, they may be so far away that when the EJC recruits the NMD factors, they don't end up binding to the ribosome. The efficiency of NMD is greatly reduced in this circumstance.
The PTC is in the first 150 bp after the coding start site. If the premature stop codon is very early, "translation reinitiation" can occur, which means translation just starts at the next Methionine codon (which is also the start codon ATC). So the premature stop codon is entirely bypassed, the ribosome translates all the way to the regular stop codon, knocking off all EJCs in the process, and the resulting protein is truncated at the beginning.
How do we use NMD Escape at GenomOncology?
GenomOncology provides automated variant interpretation algorithms for our clients. Usually nonsense or frameshift mutations in tumor suppressor genes could be interpreted as pathogenic, since the transcript will be destroyed by NMD, thus we have a loss of function. However, when a nonsense or frameshift mutation escapes NMD, we aren’t guaranteed a loss of function. The truncated protein will be created, and while this likely will still lead to a loss of function, it may have no effect, or may even lead to a gain of function! Our software will identify and clearly label any nonsense or frameshift mutations as possible NMD Escape, allowing our clients to make informed decisions for variant interpretation.
