When a person is tested for tuberous sclerosis complex (TSC), doctors look at their TSC2 gene for changes called variants that cause the disease. Many results come back labeled “variant of uncertain significance” — or VUS. That means, “We found a change in your gene, but we don’t know if it causes TSC or not.” Right now, most TSC2 variants fall into this “uncertain” category.
Figuring out each VUS one variant at a time requires slow, expensive lab tests. With over 3,600 TSC2 VUS already discovered, that approach simply cannot keep up.
Researchers Developed a Way to Test Thousands of Variants at Once
With funding from the TSC Alliance, the American Epilepsy Society, and the National Institutes of Health, a team of scientists from Northwestern University, the University of Washington, Seattle Children’s, and other institutions built a system to test thousands of variants simultaneously instead of one by one. They used two main approaches:
- Protein abundance test (VAMP-seq): They measured whether nearly 9,000 different TSC2 variants produced a normal amount of the TSC2 protein. This constitutes approximately 20% of all possible variants in the large TSC2 gene. If a variant causes too little protein to be made, it likely disrupts the gene.
- Protein activity test (SGE and cliPE): They directly measured whether 391 variants were doing their job in the cell — keeping a key growth-control switch called mTOR turned off. When TSC2 doesn’t work properly, mTOR gets stuck in the “on” position, which leads to the tumors and other problems seen in TSC.
Both Tests Together Catch More Harmful Variants
The tests showed that roughly 14–18% of the variants studied were clearly harmful. Importantly, some harmful variants do not reduce the amount of protein — they just cause the protein to stop working correctly. This means both tests are needed together to identify all disease-causing variants.
Over 75% of Uncertain Results Were Resolved
When the researchers applied their findings to a real group of patients — 276 people whose TSC2 VUS results had been sitting in the “uncertain” category — they were able to provide a definitive answer for 77% of those cases. Most (72%) were reclassified as likely harmless, and about 5% were confirmed as likely disease-causing.
This Research Has Direct Benefits for Individuals with TSC and Their Families
This work could meaningfully improve care for TSC patients in several ways:
- Clearer diagnoses. Fewer people will need to live with an uncertain genetic result.
- Access to targeted treatment. A confirmed TSC2 diagnosis opens the door to drugs called mTOR inhibitors (like everolimus or sirolimus), which directly target the disrupted pathway in TSC. Without a clear genetic result, some patients may not qualify for these therapies.
- Informed family planning. Families can more confidently understand the likelihood of passing TSC to future children.
- Reduced unnecessary worry. When a variant is reclassified as harmless, families no longer need to be concerned that it is contributing to disease.
- Clinical trial eligibility. A confirmed genetic diagnosis can qualify patients for trials of new precision therapies.
The team also noted this approach could be applied to the TSC1 gene and to other related conditions beyond TSC, potentially helping more patients who have uncertain genetic results in similar diseases. All data from this study has been made publicly available for other scientists and labs to use right away. The authors are currently seeking funding to create complete variant effect maps of both the TSC1 and TSC2 genes to vastly reduce the burden of VUS on the TSC community.
Lead author: Jeffrey D. Calhoun, PhD, Research Assistant Professor, Neurology, Feinberg School of Medicine at Northwestern University
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