Biliary-Atresia-Associated Mannosidase-1-Alpha-2 Gene Regulates Biliary and Ciliary Morphogenesis and Laterality

Abstract

Background/Aims: Infectious and genetic factors are invoked, respectively in isolated biliary atresia (BA), or syndromic BA, with major extrahepatic anomalies. However, isolated BA is also associated with minor extrahepatic gut and cardiovascular anomalies and multiple susceptibility genes, suggesting common origins.Methods: We investigated novel susceptibility genes with genome-wide association, targeted sequencing and tissue staining in BA requiring liver transplantation, independent of BA subtype. Candidate gene effects on morphogenesis, developmental pathways, and ciliogenesis, which regulates left-right patterning were investigated with zebrafish knockdown and mouse knockout models, mouse airway cell cultures, and liver transcriptome analysis.Results: Single nucleotide polymorphisms in Mannosidase-1-α-2 (MAN1A2) were significantly associated with BA and with other polymorphisms known to affect MAN1A2 expression but were not differentially enriched in either BA subtype. In zebrafish embryos, man1a2 knockdown caused poor biliary network formation, ciliary dysgenesis in Kupffer's vesicle, cardiac and liver heterotaxy, and dysregulated egfra and other developmental genes. Suboptimal man1a2 knockdown synergized with suboptimal

Mechanisms of Impaired Lung Development and Ciliation in Mannosidase-1-Alpha-2 (Man1a2) Mutants

“…We have previously shown downregulation of the ciliary genes, Ift74, and Dnah11 in the liver, and of the protein Arl13b in the lungs of untreated Man1a2 −/− mutants (So et al, 2020). Here we found that NAC exposure resulted in upregulation of all three genes in lung tissue from newborn pups of all genotypes (Supplementary Table 2).…”

“…The benefit of NAC treatment on survival likely resulted from augmented ciliogenesis as suggested by upregulation of the ciliary genes, Arl13b, Dnah11, and Ift74 in lungs of NAC-treated newborn pups with each genotype (Supplementary Table 3). We have previously reported reduced expression of these genes or corresponding proteins in Man1a2 null compared with WT newborn pups (So et al, 2020). Interestingly NAC treatment also enhanced Man1a2 exon 2 transcription in lungs from heterozygous and WT pups but not in null pups who lacked the exon 2 DNA sequence.…”

“…BA also has a complex genetic basis (Schwarz et al, 2013) and is associated with sequence variants in the ciliogenesis and planar polarity effector or the CPLANE network genes, GPC1, ADD3, ARF6, EFEMP1, and MAN1A2. These variants have modest contributions to disease in genome-wide association studies (Cheng et al, 2013;Cui et al, 2013;Ningappa et al, 2015;Chen et al, 2018;So et al, 2020). Thus, model systems for ciliary disease must incorporate the ability to study the effect of mutation burden on the phenotype and its rescue with novel strategies.…”

“…Recently, we have shown that Man1a2 −/− newborn pups which are known to develop lethal respiratory failure due to homozygous deletion of the second Man1a2 exon, also showed poor lung ciliation, and abnormal biliary epithelial proliferation (Tremblay et al, 2007;So et al, 2020). Further, several ciliary genes and pathways were dysregulated in the liver transcriptome of Man1a2 null compared with wild type (WT) newborn pups.…”

“…These pathways have a significant role in branching morphogenesis of epithelial duct networks during the development of organs like the lung, mammary gland, prostate and liver, as well as left-right patterning (Anvarian et al, 2019). Ciliation was difficult to demonstrate in liver of the Man1a2 −/− mutant mice but was visualized in respiratory epithelial cells with immunostaining of ADP Ribosylation Factor Like GTPase 13B (Arl13b), a regulatory GTPase highly enriched in cilia (Larkins et al, 2011;So et al, 2020). It is not known whether ciliation and survival were adversely affected to an intermediate level by a single copy of the heterozygous deletion in Man1a2 +/− pups, compared with WT or null pups, and whether these defects could be reversed with treatment.…”