Can High-Throughput Assays Help Classify Uncertain Variants?

The findings: Researchers designed a high-throughput functional assay that resulted in the reclassification of 26 JAG1 gene variants as likely pathogenic or causing Alagille syndrome, an inherited liver disorder that also affects the heart, eyes, bones, kidneys, vasculature, and other organs.

They assayed for membrane expression of 2,832 nucleotide variants in JAG1, a transmembrane protein-coding gene that influences gene expression. Of those gene variants, they identified 486 with abnormal membrane expression, which would be expected to inhibit normal protein function and result in Alagille syndrome. Researchers then applied this data to 144 variants of uncertain significance in patients undergoing clinical trial or research testing and were able to reclassify 26 gene variants as likely pathogenic of Alagille syndrome.

Of the 336 amino acid residues tested in the assay, 177 had at least one nucleotide change that resulted in abnormal function, with only 8 of 177 showing complete intolerance to function, where every nucleotide change at a specific amino acid residue resulted in disrupted membrane expression. The majority of residues, 169 of 177, demonstrated divergent membrane expression where only specific amino acid substitutions resulted in abnormal membrane expression. These findings indicate that loss of the wild-type amino acid does not drive pathogenicity; it is dependent on specific substitutions.

Why it matters: Advances in sequencing technologies allow researchers to identify variants of uncertain significance at unprecedented rates, and functional studies that analyze single-gene mutations have difficulty keeping up. In Alagille syndrome specifically, up to 85% of reported JAG1 missense variants have uncertain or conflicting classifications. This study uses a high-throughput functional assay to study the Alagille syndrome disease gene, JAG1, allowing for the analysis of thousands of variant effects simultaneously to help reduce uncertainty rates and the stress that comes with uncertain diagnoses.

Who conducted the study: Researchers from Children’s Hospital of Philadelphia, Perelman School of Medicine at the University of Pennsylvania, and H. Lee Moffitt Cancer Center & Research Institute conducted this study, including first and corresponding author, Melissa Gilbert, PhD, assistant professor in the Division of Genomic Diagnostics at CHOP. Tristan Hayeck, PhD, and Ramakrishnan Rajagopalan, PhD, assistant professors in the Department of Pathology and Laboratory Medicine, and senior author Nancy Spinner, PhD, chief of the Division of Genomic Diagnostics, at CHOP also contributed to the study.

How they did it: After developing a library of 2,832 JAG1 nucleotide variants, researchers designed a high-throughput membrane expression assay and identified variants that were present on the membrane (normal) from those that were absent (abnormal and a cause of Alagille syndrome). Using a large variant control set of known pathogenic and benign variants, they then calculated a weighted and normalized membrane expression score for each variant, identifying 486 variants that exhibited abnormal membrane expression. Scientists then used odds of pathogenicity calculations to correlate these scores to evidence strength levels for use during clinical variant classification. Of the 486 variants, 277 were classified as abnormal and 209 as likely abnormal, with both categories having implications for improving clinical diagnosis.

Quick thoughts: “The high-throughput assay that we’ve designed allows us to study thousands of variants simultaneously, along with a significant number of controls, providing us with functional data that helps to resolve the uncertainty of those variants,” Dr. Gilbert said. “Alagille syndrome is a multisystem disorder with variable expressivity, and molecular confirmation of a diagnosis is critical to help direct the clinical course for all children, but particularly for those who may have a milder disease presentation.”

What’s next: Future research will involve expanding high-throughput analyses. The study team recently received an R01 grant through the National Institute of Health/National Institute of Diabetes and Digestive and Kidney Diseases to design new variant libraries for both JAG1 and NOTCH2, which is also involved in Alagille syndrome pathogenesis. They plan to design assays that look at different functional features of these disease genes and test these assays in different types of cell systems. This work will further improve diagnostics and pave the way for utilization of these assays for different disease models.

Where the study was published: The study appears in the American Journal of Human Genetics.