Impact and Utilization of Scalable Functional Assays in Alagille Syndrome

PROJECT SUMMARY/ABSTRACT Uncertainty in our understanding of the functional impact of variation in the human genome has grossly impeded our ability to realize the full potential of now mature sequencing technologies. There is a universal need in genomics for the development of tools to support high-throughput functional characterization of

genomic variants in disease genes. This proposal will develop Multiplexed Assays of Variant Effects (MAVEs) to functionally assess genomic variation of over 10,000 variants in a ligand and receptor pair, JAGGED1 (JAG1) and NOTCH2, that result in the autosomal dominant disorder, Alagille syndrome, a pediatric cause of

liver, cardiac, vertebral, renal, ocular, and facial anomalies. Functional haploinsufficiency is the mechanism for JAG1-related disease and the likely mechanism for NOTCH2-related disease, which is supported by the high- frequency of protein-truncating and large gene deletions seen for JAG1-related disease and a by a high

intolerance for loss-of-function variants observed in population databases for NOTCH2. Missense variants, which are identified in 15% of patients with a JAG1 variant and >50% of patients with a NOTCH2 variant, are notoriously difficult to classify, but those that are pathogenic show defects related to protein misfolding,

intracellular retention, and an inability to heterodimerize. Moreover, a high incidence of missense variants of uncertain consequence in both genes identified during routine panel testing for general cholestatic disease confuses both patients and providers. Resolution of the functional consequence of all missense variation in

JAG1 and NOTCH2 through the development of high-throughput tools would greatly improve diagnostics and reduce reporting of non-disease-associated variants. We propose the development of MAVEs to simultaneously test the functional consequence of all disease- relevant missense variants in JAG1 and a subset of those in NOTCH2. In Aim 1 we will design two separate

MAVEs to analyze membrane expression and NOTCH2-binding ability of all JAG1 nucleotide permutations as readouts of protein function. In Aim 2 we will modify these MAVEs for use in studying all nucleotide permutations across a region of interest in NOTCH2 that contains the JAG1-binding interface. In Aim 3 we will

test the fidelity of our assay system by studying variant effects in liver cell lines, including both a high- throughput MAVEs approach and a low-throughput, but high-fidelity induced pluripotent stem cell (iPSC) model approach. Aims 1 and 2 will include complementary structural modeling and binding assays while Aim 3 will

interrogate the role of cellular environment using disease-relevant cell lines, all of which we expect will validate and extend our findings, providing insight into the biochemical and cellular consequences of pathogenic variation. Ultimately, these data will resolve uncertainty in JAG1 missense variant function that will benefit

diagnostics, improve our understanding of the functional consequence for NOTCH2 variants, and provide guidance for how to incorporate this functional data as useable evidence during clinical variant interpretation.