Termination codons in the last exon are not pathogenic without additional evidence because they have a fundamentally different effect on the protein product than termination codons found in other exons.
To understand why we need to know how the cell makes protein products from RNA and the role that termination codons usually play in that process:
First, the cell copies the DNA into an initial messenger RNA molecule that contains both exons and introns.
Next, the spliceosome complexes remove the introns leaving only the exons, with exon junction complexes (EJC) at the position of the original splice junction.
Then, the protein transcription machinery (ribosomes) starts translating the messenger RNA into protein. The process stops when the machinery reaches the termination codon. This is the signal that the protein transcription machinery uses to ‘know’ when to stop adding amino acids to the growing protein chain. Along the way, the protein transcription machinery also removes the exon-junction complexes from the RNA.
Once one copy of the protein product is made from the RNA, dozens, if not hundreds, of additional protein copies are made from that one molecule of RNA.
Sometimes, a variant creates a second termination codon earlier in the gene. This is known as a ‘premature’ terminal codon.
The RNA copy is made and spliced normally, leaving exon-junction complexes wherever splicing occurred.
In this situation, the protein transcription machinery stops when it reaches the premature termination codon instead of the original termination codon and at least one of the exon-junction complexes remains on the RNA.
Now, a different process kicks in. Because exon-junction complexes should be removed during translation, any RNA molecules that still retain exon-junction complexes must have a premature termination codon. Specialized surveillance machinery is used to find these RNA molecules.
Once the machinery finds the RNA molecules, it breaks them down so that they don’t continue to create truncated protein products.
Now that we understand how the cell makes protein products from RNA and the role of termination codons, we can conclude our original question: “Why are termination codons in the last exon reported as VUS?”.
If the premature termination codon is found within the last exon, the RNA molecule will not retain any extra EJC’s so the surveillance machinery won’t be able to identify and break it down. As a result, the RNA will continue to create a protein product, except the product will be lacking whatever residues would have been present in the full-length of the protein. So while most premature termination codons that are positioned anywhere else in the gene will lead to a nearly complete loss of the protein product, premature termination codons in the last exon are more akin to a deletion of the end of the gene. Without additional clinical or functional evidence showing that the deleted amino acids are deleterious, premature truncations in the last exon are of uncertain significance.