Identification of a Wide Spectrum of Ciliary Gene Mutations in Nonsyndromic Biliary Atresia Patients Implicates Ciliary Dysfunction as a Novel Disease Mechanism

Background: Biliary atresia (BA) is the most common obstructive cholangiopathy in neonates, often progressing to end-stage cirrhosis. BA pathogenesis is believed to be multifactorial, but the genetic contribution, especially for nonsyndromic BA (common form:>85%) remains poorly defined. Methods: We conducted whole exome sequencing on 89 nonsyndromic BA trios to identify rare variants contributing to BA etiology. Functional evaluation using patients’ liver biopsies, human cell and zebrafish models were performed. Clinical impact on respiratory system was assessed with clinical evaluation, nasal nitric oxide (nNO), high speed video analysis and transmission electron microscopy. Findings: We detected rare, deleterious de novo or biallelic variants in liver-expressed ciliary genes in 31.5% (28/89) of the BA patients. Burden test revealed 2.6-fold (odds ratio (OR) [95% confidence intervals (CI)]=2.58 [1.15-6.07], adjusted p=0.034) over-representation of rare, deleterious mutations in liver-expressed ciliary geneset in patients compared to controls. Functional analyses further demonstrated absence of cilia in the BA livers with KIF3B and TTC17 mutations, and knockdown of PCNT, KIF3B and TTC17 in human control fibroblasts resulted in reduced number of cilia. Additionally, CRISPR/Cas9-engineered zebrafish knockouts of KIF3B, PCNT and TTC17 displayed reduced biliary flow. Abnormally low level of nNO was detected in 80% (8/10) of BA patients carrying deleterious ciliary mutations, implicating the intrinsic ciliary defects. Interpretation: Our findings support strong genetic susceptibility for nonsyndromic BA. Ciliary gene mutations leading to cholangiocyte cilia malformation and dysfunction could be a key biological mechanism in BA pathogenesis. Funding Statement: This work was supported by research grants from the Hong Kong General Research Fund (grant no. 17107314 and 766112 to M.M.G.B., 17105119 to V.C.H.L.) and Li Ka Shing Donation Account - Enhanced New Staff Start-up Packages to P.H.Y.C. Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: The study protocol was approved by the Institutional Review Board of the University of Hong Kong - Hospital Authority Hong Kong West Cluster (UW 05-282 T/945). Informed consent, or informed parental consent for those under 18 years old, was obtained from all participants.