Multiple susceptibility enhancer variants increasing ADD3 expression predisposes to biliary atresia risk

Area of Science:

• Genetics
• Developmental Biology
• Hepatology

Background:

• Non-syndromic biliary atresia (BA) is a complex liver disorder with an unclear genetic basis.
• Previous studies identified 154 BA-associated single nucleotide polymorphisms (SNPs) near the ADD3 locus, particularly in Asian populations, but causal variants remain unknown.

Purpose of the Study:

• To identify the causal variants within the 154 BA-associated SNPs.
• To functionally characterize the role of ADD3 dysregulation in hepatobiliary development and BA pathogenesis.

Main Methods:

• Bioinformatic prediction of regulatory potential for 154 BA-associated SNPs.
• Functional validation using dual-luciferase reporter assays to assess enhancer activity of cis-regulatory elements (CREs).
• Zebrafish models to investigate the role of the ADD3 ortholog (add3a) in hepatobiliary development via mRNA overexpression and morpholino knockdown.

Main Results:

• 28 of 154 SNPs were located in 10 putative CREs with enhancer function; 8 showed significant enhancer activity in vitro.
• Risk haplotypes at three loci demonstrated significantly higher enhancer activity compared to non-risk haplotypes.
• Zebrafish add3a expression was observed in developing livers; its perturbation led to gallbladder abnormalities and reduced bile duct density, mimicking BA.

Conclusions:

• Multiple risk variants within enhancers upregulate ADD3 expression, contributing to biliary atresia pathogenesis.
• Genetic variants driving ADD3 upregulation are implicated in BA development.