Researchers develop new way to recognize Alagille-causing mutations
Screening system may improve diagnosis of 'this challenging disease'
A team of researchers has developed a new screening system to identify previously unrecognized mutations as the likely cause of Alagille syndrome.
In a recent study, the scientists focused on missense variants, a class of mutation for which it is often difficult to determine their functional effects on the resulting protein, and therefore whether they are disease-causing or not.
“Using a variety of methods, we developed a high-throughput assay and were able to reduce uncertainty when it comes to classifying these missense variants and improving our ability to provide a more accurate diagnosis of Alagille syndrome,” Melissa Gilbert, PhD, the study’s first author and a research assistant professor at Perelman School of Medicine of the University of Pennsylvania and Children’s Hospital of Philadelphia (CHOP), said in a CHOP press release.
The study, “Functional characterization of 2,832 JAG1 variants supports reclassification for Alagille syndrome and improves guidance for clinical variant interpretation,” was published in The American Journal of Human Genetics.
Alagille syndrome is an inherited disease that can lead to abnormalities in the liver, heart, blood vessels, bones, and other organs. Around 95% of people meeting the clinical criteria for it carry mutations in the JAG1 gene, but this does not “directly translate to diagnostic yield, as a subset of variants are classified as variants of uncertain significance (VOUSs),” the researchers wrote.
Alagille-causing mutations lead to loss of working JAG1 protein
JAG1 encodes production of the jagged 1 (JAG1) protein, which belongs to a signaling cascade that’s key for embryonic development. In general, Alagille-causing mutations lead to a loss of a working JAG1 protein.
When genetic testing identifies a JAG1 mutation in a patient, doctors still have to establish whether it’s likely to disrupt JAG1’s production or function substantially enough to cause Alagille. To do so, scientists look at several factors, including where on the gene the mutation resides and how it is expected to alter the protein.
This can be particularly difficult for missense mutations, a type of variant that results in only one amino acid (protein’s building blocks) being swapped for another in the resulting protein. While the protein’s code may be different, it is not always obvious if and how this will affect its function.
“Consequently, up to 85% of reported JAG1 missense variants have uncertain or conflicting classifications,” the researchers wrote, meaning that an Alagille diagnosis could be missed in some patients.
Now, the researchers have developed a model that could be used to better evaluate the functional significance of JAG1 missense mutations in the clinic.
They generated a library of 2,832 JAG1 mutations representing nearly all possible variants in the first seven protein-coding portions of the gene, which is a region encoding important parts of the JAG1 protein and where a larger number of Alagille-causing mutations and missense variants have been reported.
Each mutation was then evaluated based on the amount of JAG1 protein present at the cell’s surface, where it exerts its function. Impaired ability to transport JAG1 to the cell surface is “a known mechanism causing [Alagille],” the researchers wrote.
Ultimately, the researchers developed a threshold score below which the amount of cell surface JAG1 could be classified as functionally abnormal, using known harmless and harmful variants to help calibrate it.
90% of 486 abnormal mutations identified were missense variants
Based on this, they determined 486 of the nearly 3,000 evaluated mutations were abnormal or likely abnormal, 439 of which (90.3%) were missense mutations.
The researchers then applied their system to 144 variants of unknown significance that have previously been reported in clinical or research settings. They found 27 of them led to functionally abnormal JAG1, and including this information during variant reclassification allowed all but one to be reclassified as likely disease-causing.
The scientists emphasized that for other VOUSs that were not found to be functionally abnormal in their pipeline, that doesn’t mean they don’t cause disease, “as lack of [cell surface JAG1] is only one way to render JAG1 ineffective.” For now, those mutations will remain classified as VOUS.
“In the immediate future, we hope that this dataset can provide genetic counselors and clinicians with an enhanced ability of classifying these variants when they are first detected in patients with Alagille syndrome and reduce the uncertainty and anxiety faced by families with patients faced with this challenging disease,” said Nancy B. Spinner, PhD, the study’s senior author and chief of the division of genomic diagnostics at CHOP.
The team plans to continue working toward understanding the significance of all identified JAG1 mutations, as well as mutations in other genes known to cause various other diseases.
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