Biliary atresia is associated with polygenic susceptibility in ciliogenesis and planar polarity effector genes

Background & aimsBiliary atresia (BA) is poorly understood and leads to liver transplantation (LT), with the requirement for and associated risks of lifelong immunosuppression, in most children. We performed a genome-wide association study (GWAS) to determine the genetic basis of BA.MethodsWe performed a GWAS in 811 European BA cases treated with LT in US, Canadian and UK centers, and 4,654 genetically matched controls. Whole-genome sequencing of 100 cases evaluated synthetic association with rare variants. Functional studies included whole liver transcriptome analysis of 64 BA cases and perturbations in experimental models.ResultsA GWAS of common single nucleotide polymorphisms (SNPs), i.e. allele frequencies >1%, identified intronic SNPs rs6446628 in AFAP1 with genome-wide significance (p = 3.93E-8) and rs34599046 in TUSC3 at sub-threshold genome-wide significance (p = 1.34E-7), both supported by credible peaks of neighboring SNPs. Like other previously reported BA-associated genes, AFAP1 and TUSC3 are ciliogenesis and planar polarity effectors (CPLANE). In gene-set-based GWAS, BA was associated with 6,005 SNPs in 102 CPLANE genes (p = 5.84E-15). Compared with non-CPLANE genes, more CPLANE genes harbored rare variants (allele frequency <1%) that were assigned Human Phenotype Ontology terms related to hepatobiliary anomalies by predictive algorithms, 87% vs. 40%, p <0.0001. Rare variants were present in multiple genes distinct from those with BA-associated common variants in most BA cases. AFAP1 and TUSC3 knockdown blocked ciliogenesis in mouse tracheal cells. Inhibition of ciliogenesis caused biliary dysgenesis in zebrafish. AFAP1 and TUSC3 were expressed in fetal liver organoids, as well as fetal and BA livers, but not in normal or disease-control livers. Integrative analysis of BA-associated variants and liver transcripts revealed abnormal vasculogenesis and epithelial tube formation, explaining portal vein anomalies that co-exist with BA.ConclusionsBA is associated with polygenic susceptibility in CPLANE genes. Rare variants contribute to polygenic risk in vulnerable pathways via unique genes.Impact and implicationsLiver transplantation is needed to cure most children born with biliary atresia, a poorly understood rare disease. Transplant immunosuppression increases the likelihood of life-threatening infections and cancers. To improve care by preventing this disease and its progression to transplantation, we examined its genetic basis. We find that this disease is associated with both common and rare mutations in highly specialized genes which maintain normal communication and movement of cells, and their organization into bile ducts and blood vessels during early development of the human embryo. Because defects in these genes also cause other birth defects, our findings could lead to preventive strategies to lower the incidence of biliary atresia and potentially other birth defects

The role of dynamic DNA methylation in liver transplant rejection in children

BackgroundTranscriptional regulation of liver transplant (LT) rejection may reveal novel predictive and therapeutic targets.PurposeTo test the role of differential DNA methylation in children with biopsy-proven acute cellular rejection (rejectors, R) after LT.MethodsPaired peripheral blood DNA samples were obtained before and after LT from 17 children, including 4R and 13 non-rejector (NR), and assayed with MethylC capture sequencing (MCC-Seq) approach covering 5 million CpGs in immune-cell specific regulatory elements. Differentially methylated CpGs (DMCs) were identified using generalized linear regression models adjusting for sex and age and merged into differentially methylated regions (DMR) comprising 3 or more DMCs.ResultsContrasting R vs NR, we identified 2238 DMCs in post-LT and 2620 DMCs in pre-LT samples, which clustered in 216 and 282 DMRs respectively. DMCs associated with R were enriched in enhancers and depleted in promoters. The proportion of hypomethylated versus hypermethylated DMRs increased from 22% to 48% (p&lt;0.0001) in pre-LT vs. post-LT DMCs, respectively. The highest-ranked biological processes enriched in post-LT DMCs were antigen processing and presentation via MHC class I, MHC class I complex, and peptide binding (p&lt;7.92E-17), respectively. Top-ranked DMRs mapped to genes which mediate B-cell receptor signaling (ADAP1) or regulate several immune cells(ARRB2)(p&lt;3.75E-08). DMRs in MHC class I genes were enriched for SNPs which bind TFs, affect gene expression and splicing, or alter peptide-binding amino acid sequences.ConclusionsDynamic methylation in distal regulatory regions reveals known transplant-relevant MHC-dependent rejection pathways, and identifies novel loci for future mechanistic evaluations in pediatric transplant subcohorts.</jats:sec

T-cell and antibody immunity after COVID-19 mRNA vaccines in healthy and immunocompromised subjects-An exploratory study

AbstractBackgroundIn recent studies, up to half of immunocompromised (IC) subject populations fail to develop antibodies after COVID-19 vaccination.Purpose and MethodsHere, we explore whether T-cells which respond to the spike (S) antigenic sequence and its less conserved S1, and the conserved S2 component are present in serial samples before and after each dose of mRNA1273 or BNT162b2 vaccines in 20 healthy immunocompetent subjects. Single samples from 7 vaccinated IC subjects were also tested. Simultaneously, we measured IgG antibodies to the receptor binding domain (RBD) of S1, and anti-S IgG, and frequencies of monocytic CD14+HLA-DR-(M-MDSC) and polymorphonuclear CD14-CD15+CD11b+ (PMN-MDSC) myeloid-derived suppressor cells.ResultsIn healthy subjects, S1-, S2-, and S-reactive CD4 and CD8 T-cell frequencies showed a numeric but not statistically significant decrease after the first vaccine dose and were accompanied by increased MDSC frequencies (p&lt;0.05). After the second dose, S2-and S-reactive CD4 and CD8 cells and MDSC approached pre-vaccination levels. In healthy subjects, a) S1-reactive CD8 frequencies were significantly higher after the second dose compared with pre-vaccination levels (p=0.015), b) anti-RBD and anti-S IgG were present in all after the second dose. Among seven IC subjects, anti-RBD and anti-S IgG were absent in 4 and 3 subjects, respectively. S1-reactive CD8 cells were identified in 2 of 4 anti-RBD negative subjects. S-reactive CD4 or CD8 cells were identified in all three anti-S negative subjects.ConclusionsIn healthy immunocompetent subjects, mRNA vaccines induce antibodies to the spike antigenic sequences and augment CD8 cells reactive to the S1 spike sequence, which is more specific for the SARS-CoV-2 virus. In this exploratory cohort of vaccinated immunocompromised subjects, S1-reactive CD8 cells can be detected in some who are negative for RBD antibody, and S-reactive T-cells are present in all who are negative for spike antibody.</jats:sec

Immune Response in Severe and Non-Severe Coronavirus Disease 2019 (COVID-19) Infection: A Mechanistic Landscape

The mechanisms underlying the immune remodeling and severity response in coronavirus disease 2019 (COVID-19) are yet to be fully elucidated. Our comprehensive integrative analyses of single-cell RNA sequencing (scRNAseq) data from four published studies, in patients with mild/moderate and severe infections, indicate a robust expansion and mobilization of the innate immune response and highlight mechanisms by which low-density neutrophils and megakaryocytes play a crucial role in the cross talk between lymphoid and myeloid lineages. We also document a marked reduction of several lymphoid cell types, particularly natural killer cells, mucosal-associated invariant T (MAIT) cells, and gamma-delta T (γδT) cells, and a robust expansion and extensive heterogeneity within plasmablasts, especially in severe COVID-19 patients. We confirm the changes in cellular abundances for certain immune cell types within a new patient cohort. While the cellular heterogeneity in COVID-19 extends across cells in both lineages, we consistently observe certain subsets respond more potently to interferon type I (IFN-I) and display increased cellular abundances across the spectrum of severity, as compared with healthy subjects. However, we identify these expanded subsets to have a more muted response to IFN-I within severe disease compared to non-severe disease. Our analyses further highlight an increased aggregation potential of the myeloid subsets, particularly monocytes, in COVID-19. Finally, we provide detailed mechanistic insights into the interaction between lymphoid and myeloid lineages, which contributes to the multisystemic phenotype of COVID-19, distinguishing severe from non-severe responses.</jats:p