Integrated RNA-based functional characterization facilitates reclassification of BRCA1/2 variants of uncertain significance in a clinical cancer cohort

Every cell in the human body carries a set of instructions, written in a chemical code called DNA, that tells it how to build and maintain itself. Among these instructions are specific genes that act as guardians, constantly repairing damage to the genetic code to prevent cells from growing out of control and becoming cancer. Two of the most important guardians are genes named BRCA1 and BRCA2. When these genes work correctly, they help fix broken strands of DNA. But when they are damaged by a change in their sequence, known as a variant, the repair system fails, and the risk of developing breast, ovarian, and other cancers rises significantly.

For doctors and patients, knowing the status of these genes is a powerful tool. It can guide decisions about screening, prevention, and treatment. However, the story is not always simple. Genetic testing often reveals changes in the DNA sequence that have never been seen before or whose effects are unknown. These are called variants of uncertain significance. They are genetic questions marks. A doctor cannot tell a patient if such a change is harmless or dangerous based on the DNA sequence alone. This uncertainty leaves families in a difficult position, unable to make fully informed choices about their health. The challenge is to figure out what these mysterious changes actually do inside the cell.

A team of researchers at Zhongshan Hospital in Shanghai decided to tackle this problem by looking at the next step in the genetic process: how the DNA instructions are read and copied into RNA. In the cell, DNA is first transcribed into a molecule called RNA, which then serves as a template for building proteins. Before this RNA can be used, it must be edited. Imagine a long sentence with several clauses; the cell must cut out the unnecessary parts and stitch the important ones together to form a clear message. This editing process is called splicing. If a genetic variant disrupts this editing, the final message becomes garbled, and the resulting protein may be broken or missing entirely. The researchers suspected that many of the uncertain variants they encountered were causing these splicing errors, but the computer programs used to predict such errors were often unreliable or gave conflicting answers.

To get a definitive answer, the researchers started with a large group of 1,762 patients who had undergone genetic testing for hereditary cancer. From this group, they identified 60 unique variants of uncertain significance in the BRCA1 and BRCA2 genes. They knew they could not test all of them in living patients, so they used a clever laboratory strategy. They took small pieces of DNA containing the specific regions where the variants occurred and inserted them into a simple, artificial system inside human cells grown in a dish. This system, known as a minigene, acted as a miniature factory that would produce RNA based on the instructions in the inserted DNA. By watching what RNA came out of this factory, the scientists could see exactly how the cell processed the genetic instructions.

They selected five of the most promising candidates for this experiment, focusing on those that computer models suggested might cause splicing problems. The results were revealing. Two of the variants, both located in the BRCA2 gene, caused the cell to make mistakes in how it stitched the RNA together. One of these changes caused the cell to skip an entire section of the instructions, while the other caused a more complex error where the cell skipped a section and also grabbed onto the wrong starting points nearby. These mistakes resulted in RNA messages that were missing critical pieces of information. The other three variants they tested did not cause any errors; the cells processed them just as they would have processed normal DNA.

The researchers did not stop at simply seeing that errors occurred. They wanted to understand the full shape of the mistake. Using a technique called RNA sequencing, they read the exact sequence of the faulty RNA molecules produced by the two problematic variants. This confirmed that the errors were real and detailed exactly which parts of the message were missing or added. For the variant that caused a skip, the missing section corresponded to a stretch of 52 building blocks in the final protein. To understand what this loss meant, the team used powerful computer simulations to model the structure of the BRCA2 protein. They found that the missing section was located in a critical area where the protein interacts with another molecule to perform its repair work. While the simulation could not prove the protein was completely broken, it showed that the missing piece came from a tightly organized and essential part of the structure, suggesting the protein would likely fail to function properly.

The final piece of the puzzle came from looking at the families of the patients. The researchers traced the genetic history of the two variants that caused errors. They found that these changes appeared in multiple family members, some of whom had developed cancer and others who had not. While this pattern was not a perfect match for a disease that always strikes, it provided supporting evidence that these variants were indeed linked to the disease. By combining the laboratory proof of the splicing errors, the computer models of the protein structure, and the family history, the team was able to reclassify the two harmful variants. They moved them from the category of "uncertain" to "likely pathogenic" and "pathogenic," giving doctors and patients a clear answer. The three variants that caused no errors were reclassified as likely harmless.

This study demonstrates that when genetic testing leaves a question mark, looking at how the cell actually reads the DNA can provide the answer. The researchers showed that computer predictions alone are often not enough to solve these mysteries because the cell's editing machinery can behave in complex and unexpected ways. A single change in the DNA can lead to multiple different types of errors, or it might have no effect at all. By using a combination of lab experiments, detailed sequencing, and structural modeling, the team was able to cut through the uncertainty. Their work offers a practical path forward for resolving similar genetic questions, turning vague risks into clear medical guidance for families facing the threat of hereditary cancer.