# Resolving Uncertainty in Alagille Syndrome Diagnostics

> **NIH NIH R01** · CHILDREN'S HOSP OF PHILADELPHIA · 2024 · $591,736

## Abstract

PROJECT SUMMARY/ABSTRACT
Uncertainty in genomic diagnostics creates a barrier to realizing the full potential of genomic medicine.
Uncertainty is evident in: 1) our inability to determine if some DNA variants are pathogenic or benign and 2) our
inability to predict to what extent a person with a disease-causing variant will be affected, due to variable
expressivity. This proposal will study both phenomena for the autosomal dominant disorder Alagille syndrome,
caused by mutations in one of two genes in the Notch signaling pathway, the ligand JAGGED1 (JAG1) or the
receptor, NOTCH2. Alagille syndrome is characterized by pediatric liver, heart, vertebral, renal, ocular, and
facial anomalies with highly variable expressivity. The mechanism of disease for JAG1-related Alagille
syndrome is haploinsufficiency whereas the mechanism for NOTCH2-related Alagille syndrome is less clear,
with fewer reported variants, less functional characterization, and a higher prevalence of missense variants
(>50%). Missense variants are difficult to classify, often requiring functional validation to support or reject
pathogenicity. In Alagille syndrome, functional characterization has been carried out for only 19/125 reported
missense mutations, thus, despite a high detection rate, the diagnostic rate is lower due to this uncertainty.
We propose to resolve uncertainty in the diagnostics of Alagille syndrome using assays designed to
characterize the pathogenicity of JAG1 and NOTCH2 missense variants and analysis of gene expression data
from patient liver samples to identify gene expression signatures that can be used for genotype-phenotype
evaluations. In Aim 1, we will design a Site Saturation Variant Library of all possible nucleotide permutations at
each nucleotide position across a region with high missense variant uncertainty in the JAG1 C-terminus and
test this library by developing a Multiplexed Assay for Variant Effects (MAVEs) that will measure cellular
localization of JAG1 as a readout of protein function. In Aim 2, we will use FFPE liver tissue samples to
analyze gene expression differences between Alagille syndrome patients and controls, as well as between
Alagille syndrome patients with mild versus severe liver disease. In Aim 3, we will study the molecular basis of
NOTCH2 variants through functional, expression, and enzymatic assays using mutant cell lines. We
hypothesize that these proposed assays will identify a high-throughput method to test missense pathogenicity
(Aim 1), identify gene expression differences between Alagille syndrome patients and controls as well as gene
expression signatures that are different between Alagille syndrome patients with mild versus severe liver
disease (Aim 2), and determine the mechanism by which NOTCH2 variants cause Alagille syndrome through
functional analysis (Aim 3). Ultimately, these data will improve variant analysis for Alagille syndrome,
improve our understanding of the molecular basis of liver disease in Alagille s...

## Key facts

- **NIH application ID:** 10861034
- **Project number:** 5R01DK134585-02
- **Recipient organization:** CHILDREN'S HOSP OF PHILADELPHIA
- **Principal Investigator:** Nancy Bettina Spinner
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $591,736
- **Award type:** 5
- **Project period:** 2023-07-01 → 2028-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10861034

## Citation

> US National Institutes of Health, RePORTER application 10861034, Resolving Uncertainty in Alagille Syndrome Diagnostics (5R01DK134585-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10861034. Licensed CC0.

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