A recent Northwestern Medicine study offers new data showing previously unknown genetic variants that cause the development of tuberous sclerosis complex, a rare genetic disorder that causes benign tumors to develop in many parts of the body, according to findings published in Nature Communications.
Jeffrey Calhoun, PhD, research assistant professor in the Ken and Ruth Davee Department of Neurology’s Division of Epilepsy and Clinical Neurophysiology, was senior author of the study.
Tuberous sclerosis complex (TSC) tumors are most commonly observed in the skin, brain, eyes, kidneys, heart and lungs. Symptoms and symptom severity can vary among patients, but may include changes in the skin, seizures, behavioral issues and heart and lung problems, among others.
Genetic tests, electroencephalogram, brain magnetic resonance imaging and a physical exam are often used to diagnose TSC. While there is no cure for TSC, precision treatment strategies can help manage specific symptoms as they arise.
TSC is caused by genetic variants in two mTOR pathway genes: TSC1 and TSC2. When either gene functions abnormally, mTOR pathway activity is elevated, leading to pathology.
Current functional assays, however, have been unable to determine the significance of thousands of already observed TSC2 missense variants. Missense variants are changes in DNA coding for the TSC2 protein, which cause a single amino acid among over 1,800 to be substituted for a different amino acid.
“Missense variants in TSC2 are often classified as variants of uncertain significance, which act as barriers to a precise genetic diagnosis of TSC. A precise genetic diagnosis can be valuable to enable access to precision treatment with mTOR pathway inhibitors,” Calhoun said.
To address this challenge, Calhoun’s team used next-generation sequencing to measure the steady-state abundance, or the stable level of proteins created, of nearly 9,000 TSC2 missense variants.
Next, the scientists developed an mTOR pathway activity assay using genome editing and cell sorting based on an mTOR pathway biomarker to create activity scores for 391 missense variants, Calhoun said.
“Say we have 10,000 cells. Each of those cells has a different missense variant, and then we can sort those cells based on how active the mTOR pathway is. If it is highly active, those cells are likely to harbor a pathogenic variant that causes TSC. If instead the cell exhibits normal levels of mTOR activity, those genetic variants are likely to be benign,” Calhoun said.
Of the total missense variants that were assayed, the scientists found that 14 percent had altered TSC2 abundance and nearly 18 percent had altered mTOR pathway activity.
Overall, the data reclassified 212 of 276, or 78.8 percent, TSC2 missense variants of uncertain significance.
The new data could help improve genetic diagnosis of TSC and improve patient care through proper surveillance, family planning and access to precision therapies. The data may also help enable patients to enroll in gene replacement clinical trials, during which individuals receive healthy copies of either TSC1 or TSC2
Calhoun said his team now aims to scale all missense variants in the TSC2 gene and eventually the TSC1 gene, as well.
“There are many individuals who have variants of uncertain significance reported in TSC2. Clinicians think they might have tuberous sclerosis complex, but they don’t have definitive proof. We think this data can give more definitive proof that this variant either is or isn’t causing the disease,” Calhoun said.
“Our amazing collaborators at the University of Washington, Dr. Doug Fowler and Dr. Rich James, and the talented scientists in their laboratories made significant contributions to this work. Their work, and ours, will have a lasting contribution to the TSC community,” Calhoun added.
Carina Biar, a former Northwestern undergraduate student, was a co-first author of the study. Gemma Carvill, PhD, associate professor of Neurology in the Division of Epilepsy and Clinical Neurophysiology, was a co-author of the study.
This work was supported by an American Epilepsy Society Junior Investigator Award and a TSC Alliance Research Grant, the National Institutes of Health National Human Genome Research Institute IGVF Program (HG011969), and in part by National Human Genome Research Institute Interdisciplinary Training in Genome Sciences Grant T32HG000035.
