How variants outside known protein domains challenge our understanding of cancer risk
Imagine your body contains a remarkable security system that constantly patrols your cells, repairing damaged DNA before it can cause problems. This isn't science fiction—it's the work of the BRCA1 protein, one of our body's most crucial cancer-fighting molecules. When this cellular guardian works properly, it helps prevent tumors from developing. But when it contains errors, the risk of breast, ovarian, and other cancers increases dramatically 2 .
BRCA1 plays a critical role in fixing DNA double-strand breaks, preventing mutations from accumulating in our cells.
Harmful BRCA1 variants can increase lifetime breast cancer risk up to 72% and ovarian cancer risk up to 44%.
For decades, scientists have focused their attention on specific regions of the BRCA1 protein known as "domains"—the RING, BRCT, and coiled-coil regions—which act as critical functional hubs. Most known cancer-causing mutations occur in these areas. But what about the rest of the protein? The extensive regions between these domains have been largely overlooked, often dismissed as genetic "dark matter" without important functions. This assumption has now been challenged by groundbreaking research that reveals surprising findings in these neglected areas 1 4 .
A recent study has turned this assumption on its head, discovering that certain variants located outside the well-established domains can indeed disrupt the BRCA1 protein's stability and function. These findings could reshape how we interpret genetic test results and assess cancer risk for countless individuals and families 1 .
To appreciate these discoveries, we first need to understand BRCA1's structure. The BRCA1 protein is a massive molecule consisting of 1,863 amino acids, making it one of the larger proteins in our bodies 2 . Think of it as a sophisticated multi-tool with several specialized attachments:
Located at the beginning of the protein, this region acts as a molecular handshake that allows BRCA1 to partner with another protein called BARD1. Together, they form a crucial complex that tags damaged proteins for destruction—a process called ubiquitination 2 .
Positioned at the opposite end of the protein, these regions serve as molecular docking stations that recognize and bind to other proteins involved in DNA damage repair 2 .
This middle section facilitates BRCA1's interaction with PALB2, another important protein in the DNA repair network 4 .
For years, the extensive regions between these domains—particularly the large central section comprising approximately 1,500 amino acids—were considered mere "spacers" without critical functions. This led to the widespread assumption that most mutations in these areas were likely harmless 4 .
| Domain Name | Location in Protein | Primary Function | Known Associated Risks |
|---|---|---|---|
| RING Domain | Amino acids 22-64 | Forms complex with BARD1, provides E3 ubiquitin ligase activity | High cancer risk when mutated |
| Nuclear Localization Signals | Amino acids 503-508 & 607-614 | Directs protein to cell nucleus | Altered function when impaired |
| Coiled-coil Domain | Amino acids 1364-1437 | Binds to PALB2 protein | Interrupted protein partnerships |
| BRCT Domains | Amino acids 1646-1736 & 1760-1855 | Protein recruitment for DNA damage repair | Significant cancer risk when mutated |
As genetic testing becomes more accessible and comprehensive, laboratories are discovering an increasing number of rare genetic variants in the BRCA1 gene. Many of these are missense variants—single-letter changes in the genetic code that result in one amino acid being substituted for another in the protein 1 .
The problem? For most of these rare variants, we don't have enough information to know whether they increase cancer risk. These are classified as "Variants of Uncertain Significance" or VUS 6 . For patients and their doctors, receiving a VUS result can be frustrating and anxiety-provoking—they know there's a genetic difference, but nobody can tell them what it means for their health 4 .
Compounding this challenge is a long-standing assumption in genetics: variants located outside known functional domains are probably benign. This concept has even been formalized in some professional guidelines, which suggest that a variant's location outside key domains can be used as evidence to classify it as likely harmless 4 . But is this assumption always correct?
Only variants in known domains (RING, BRCT, coiled-coil) are considered potentially harmful. Variants elsewhere are assumed to be benign.
Research shows some variants outside known domains can disrupt protein stability and function, challenging the traditional view.
To test this assumption, a research team embarked on a comprehensive study of 14 rare BRCA1 missense variants, 13 of which were located outside the well-established domains 1 4 . Their approach was both innovative and meticulous:
The researchers chose variants identified in the "BRCA1 Norway" study—a collection of BRCA1 variants detected in Norwegian families with suspected hereditary breast and ovarian cancer. All selected variants were classified as VUS, meaning their clinical significance was unknown 4 .
Using genetic engineering techniques, the researchers introduced each of these rare variants into the full-length BRCA1 gene—a critical improvement over previous studies that often used only fragments of the gene. This allowed them to study the variants in the context of the complete protein, better mimicking the real-world situation in human cells 4 .
The team then performed a battery of tests to assess how each variant affected the BRCA1 protein:
This multi-faceted approach was crucial because BRCA1 is involved in multiple cellular processes, and a variant might affect one function while leaving others intact.
| Research Tool | Function in BRCA1 Research | Specific Application in This Study |
|---|---|---|
| pDEST-mCherry-LacR-BRCA1 plasmid | Engineered DNA construct expressing full-length BRCA1 | Served as template for introducing variants via site-directed mutagenesis |
| HEK293FT cells | Human embryonic kidney cell line | Used for protein expression analysis due to efficient transfection |
| Site-directed mutagenesis kit | Introduces specific genetic changes into DNA constructs | Created the 14 BRCA1 VUS for functional testing |
| Anti-BRCA1 antibodies | Proteins that bind specifically to BRCA1 for detection | Enabled visualization and quantification of BRCA1 in western blot assays |
| Proteasome inhibitors | Chemicals that block the cell's protein degradation machinery | Helped determine if reduced BRCA1 levels were due to increased degradation |
| RIPA buffer | Solution that breaks open cells and dissolves proteins | Used to extract proteins from cells for stability and expression studies |
The results challenged conventional wisdom. While most of the 14 variants did indeed appear to have minimal functional impact, five variants stood out as causing clear problems—and all five were located outside the well-known domains 1 4 .
Specifically, the researchers found that three variants—p.Met297Val, p.Asp1152Asn, and p.Leu52Phe—made the BRCA1 protein more prone to degradation by the cell's protein-disposal system (the proteasome). Two additional variants—p.Leu1439Phe and p.Gly890Arg—also significantly reduced protein stability compared to the normal BRCA1 protein 1 4 .
These findings demonstrated for the first time that variants in these neglected regions could indeed disrupt fundamental properties of the BRCA1 protein, potentially compromising its ability to prevent tumor development.
| Variant Name | Location Relative to Known Domains | Observed Functional Impact | Potential Clinical Significance |
|---|---|---|---|
| p.Leu52Phe | Within RING domain | Increased proteasome-mediated degradation | Likely pathogenic |
| p.Met297Val | Outside known domains | Increased proteasome-mediated degradation | Further evidence needed |
| p.Asp1152Asn | Outside known domains | Increased proteasome-mediated degradation | Further evidence needed |
| p.Leu1439Phe | Outside known domains | Reduced protein stability | Further evidence needed |
| p.Gly890Arg | Outside known domains | Reduced protein stability | Further evidence needed |
5 out of 14 tested variants showed functional impacts, all located outside established domains.
7 variants could be reclassified from VUS to "likely benign" based on functional evidence.
These findings extend far beyond basic science—they have real-world implications for how we assess cancer risk and guide medical decisions. The discovery that variants outside established domains can affect protein function suggests we need to reevaluate our approach to variant classification 1 4 .
Based on their findings, the research team suggested that seven variants could be reclassified from "uncertain significance" to "likely benign," potentially providing clarity for individuals carrying these variants. However, they also demonstrated that the blanket assumption that all variants outside key domains are harmless is flawed 1 4 .
This research highlights the importance of using multiple complementary assays that test different aspects of protein function when assessing VUS. Relying on a single test or focusing exclusively on known domains could cause us to miss important functional impacts 6 .
As genetic testing continues to evolve, studies like this remind us that there's still much to learn about the intricate workings of our genome. The "dark matter" of BRCA1—and other cancer susceptibility genes—may hold secrets that could reshape our understanding of cancer risk 1 4 .
What remains clear is that as we continue to explore the vast landscape of our genetic code, we must approach it with humility and curiosity, recognizing that nature often reserves its most surprising revelations for those willing to question established assumptions and venture into uncharted territory.