New lab-grown cells may improve Alagille syndrome variant testing
Approach detected Notch signaling changes missed in standard lab-grown cells
Written by |
A new type of genetically engineered lab-grown cell may help researchers determine whether certain genetic variants can contribute to Alagille syndrome, a study reports.
Certain mutations in the JAG1 and NOTCH2 genes can cause Alagille by disrupting a signaling pathway called Notch. However, not all variants in these genes cause the disease, and many are classified as variants of unknown significance (VUSs) because researchers aren’t certain whether they contribute to disease-related problems.
To help classify VUSs, the research team developed cells that could more sensitively detect variant-related changes in JAG1-NOTCH2 signaling activity.
New cell model may improve Alagille variant testing
“Our analyses … allow for a more sensitive comprehension of the JAG1 and NOTCH2 variants impact on the Notch signaling pathway activity and, thus, an improved interpretation and classification of variants involved in ALGS [Alagille syndrome],” the researchers wrote.
The study, “Improved functional JAG1 and NOTCH2 variant testing in patients with clinical or suspected Alagille syndrome using new low-Notch activity cells,” was published in Human Genetics.
The Notch signaling pathway plays important roles in embryonic development, and certain mutations in one of the copies of the JAG1 or NOTCH2 genes can disrupt this pathway and cause Alagille. This genetic disease is marked by problems in the liver and heart, but it can affect several other body systems.
Because symptoms can vary in nature and severity, it can be difficult to classify a genetic variant as disease-causing or benign based on clinical features alone. Testing a variant’s effects on Notch signaling activity in lab-grown cells may help reclassify VUSs. In these tests, researchers introduce genetic material carrying the variant into cells and measure how it affects Notch signaling activity.
Researchers would expect disease-causing variants to decrease Notch activity in these types of tests. However, background Notch signaling in lab-grown cells may obscure these changes, making it more difficult to assess the effects.
Low-Notch cells designed to reveal variant effects
As a potential solution to this problem, the researchers genetically engineered human cells to have very low Notch activity. These cells, called HEK293T low Notch activity (HEK293T-LNA) cells, were designed to make it easier for researchers to evaluate the effects of JAG1 or NOTCH2 variants.
“The HEK293T-LNA cells thus allowed us to specifically test the signaling potential of the respective JAG1-NOTCH2 variants in a low-Notch activity context,” the team wrote.
The researchers tested the cells’ utility in nine individuals with clinical or suspected Alagille who carried a variant in one copy of the JAG1 or NOTCH2 gene. These participants had differing clinical features — some clearly met diagnostic criteria for Alagille while others had isolated or atypical disease features.
A total of eight different variants were identified and classified as VUSs at the beginning of the study. The researchers also evaluated the effects of known disease-causing variants in JAG1 and NOTCH2, as well as benign variants and normal versions of the genes.
The team first conducted the tests in standard lab-grown cells. Known disease-causing NOTCH2 variants significantly decreased Notch signaling activity relative to benign variants or the normal version of the gene.
However, known disease-causing JAG1 variants didn’t significantly alter signaling in the standard cells, which prompted the team to hypothesize that background Notch activity was obscuring changes related to the variants.
When the team conducted the same tests in the newly created HEK293T-LNA cells, they found that normal versions of the JAG1 and NOTCH2 genes increased Notch signaling, as expected for cells with low Notch activity. By comparison, known disease-causing variants in each gene significantly decreased Notch signaling.
Two variants tied to reduced Notch signaling
Among VUSs, only two of the eight variants — p.Cys412Phe in the NOTCH2 gene and p.Leu18Arg in the JAG1 gene — were associated with reduced Notch signaling. This allowed p.Cys412Phe to be reclassified from VUS to likely disease-causing, even though the patient carrying this variant didn’t show clear clinical signs of Alagille.
“The patient is currently clinically asymptomatic with a mild liver [clinical profile] … underlining the highly variable expressivity in ALGS,” the team wrote.
The Notch signaling reduction seen with p.Leu18Arg, which earlier research had suggested was likely disease-causing, was less pronounced than with well-established disease-causing variants.
This could suggest that the variant affected JAG1 protein stability or processing rather than directly suppressing signaling, according to the team. A follow-up experiment supported this hypothesis. Based on this, and the fact that the corresponding patient had clear clinical signs of Alagille, the team said the findings supported the JAG1 VUS as likely disease-causing.
“Our experiment serves as an independent validation [of that high-throughput approach],” the team wrote.
The study demonstrates that using HEK293T-LNA cells can help reveal Notch signaling problems. However, the system can’t measure every potential disease-causing, or pathogenic, effect.
“We can therefore conclude that our functional analyses enable reclassification of a VUS as likely pathogenic if a pathogenic effect is detected, but it does not allow a VUS to be classified as likely benign if our assays show no pathogenic effect,” the researchers wrote.
Future work may continue testing the cells and their sensitivity to different types of changes in Notch signaling.
