Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1000 cases of unexplained pediatric hepatitis in children have been reported worldwide, including 278 cases in the UK. Here we report investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator subjects, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in liver, blood, plasma or stool from 27/28 cases. We found low levels of Adenovirus (HAdV) and Human Herpesvirus 6B (HHV-6B), in 23/31 and 16/23 respectively of the cases tested. In contrast, AAV2 was infrequently detected at low titre in blood or liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T-cells and B-lineage cells. Proteomic comparison of liver tissue from cases and healthy controls, identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and in severe cases HHV-6B, may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1000 cases of unexplained pediatric hepatitis in children have been reported worldwide, including 278 cases in the UK 1. Here we report investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator subjects, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in liver, blood, plasma or stool from 27/28 cases. We found low levels of Adenovirus (HAdV) and Human Herpesvirus 6B (HHV-6B), in 23/31 and 16/23 respectively of the cases tested. In contrast, AAV2 was infrequently detected at low titre in blood or liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T-cells and B-lineage cells. Proteomic comparison of liver tissue from cases and healthy controls, identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and in severe cases HHV-6B, may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

Optimisation of Delivery of the Ataxia Telangiectasia Mutated Transgene to HeLa Cells Using Glycosaminoglycan Enhanced Transduction

Ataxia telangiectasia (AT) is a monogenic syndrome that is characterised by hypersensitivity to ionising irradiation. This study is part of a proof-of-concept project for a non-viral gene therapy that utilises glycosaminoglycan-enhanced transduction (GET), which is based on transfection peptides that fuse membrane docking and cell penetrating domains. Previous work has demonstrated delivery of plasmid DNA (pDNA) encoding the AT-mutated gene (ATM) in vitro using the GET peptides FLR and FLH. The aim of this study is to optimise the GET formulation in vitro for delivery and expression of ATM pDNA in HeLa cells. Attempts were made to capture transgene expression via Zoanthus sp. green fluorescent protein (zsGreen) fluorescence and immunostaining, and to visualise results by flow cytometry and confocal microscopy. Results suggest that transfection efficiency may positively correlate with peptide : pDNA mass ratio (PDMR). They also suggest that the proteosome inhibitor MG132 may better stabilise ATM expression than the histone deacetylase inhibitor suberanilohydroxamic acid (SAHA). These results were found by flow cytometry for zsGreen fluorescence. Microscopic results due to experimental factors were inconsistent with those of flow cytometry, whereas immunostaining analysis was precluded by nonspecific binding of the secondary antibody. Further optimisation requires reproducing results for statistical analysis, validating ATM expression by immunostaining and microscopy, and replicating transfection results in ATM-/- fibroblasts. Future directions include establishing functionality of transfected ATM using irradiation assays, exploring effects of genomic integration on ATM expression, and developing formulations for in vivo performance using animal models

Optimisation of Delivery of the Ataxia Telangiectasia Mutated Transgene to HeLa Cells Using Glycosaminoglycan Enhanced Transduction

Ataxia telangiectasia (AT) is a monogenic syndrome that is characterised by hypersensitivity to ionising irradiation. This study is part of a proof-of-concept project for a non-viral gene therapy that utilises glycosaminoglycan-enhanced transduction (GET), which is based on transfection peptides that fuse membrane docking and cell penetrating domains. Previous work has demonstrated delivery of plasmid DNA (pDNA) encoding the AT-mutated gene (ATM) in vitro using the GET peptides FLR and FLH. The aim of this study is to optimise the GET formulation in vitro for delivery and expression of ATM pDNA in HeLa cells. Attempts were made to capture transgene expression via Zoanthus sp. green fluorescent protein (zsGreen) fluorescence and immunostaining, and to visualise results by flow cytometry and confocal microscopy. Results suggest that transfection efficiency may positively correlate with peptide : pDNA mass ratio (PDMR). They also suggest that the proteosome inhibitor MG132 may better stabilise ATM expression than the histone deacetylase inhibitor suberanilohydroxamic acid (SAHA). These results were found by flow cytometry for zsGreen fluorescence. Microscopic results due to experimental factors were inconsistent with those of flow cytometry, whereas immunostaining analysis was precluded by nonspecific binding of the secondary antibody. Further optimisation requires reproducing results for statistical analysis, validating ATM expression by immunostaining and microscopy, and replicating transfection results in ATM-/- fibroblasts. Future directions include establishing functionality of transfected ATM using irradiation assays, exploring effects of genomic integration on ATM expression, and developing formulations for in vivo performance using animal models

Genomic investigations of unexplained acute hepatitis in children

Funding: UKHSA funded the metagenomics and HAdV sequencing. The work was part funded by the NIHR Blood and Transplant Research Unit in Genomics to Enhance Microbiology Screening (GEMS), the National Institute for Health and Care Research (CO-CIN-01) or jointly by NIHR and UK Research and Innovation (CV220-169, MC_PC_19059). S. Morfopoulou is funded by a W.T. Henry Wellcome fellowship (206478/Z/17/Z). S.B. and O.E.T.M. are funded by the NIHR Blood and Transplant Research Unit (GEMS). M.M.M. and M.L. are supported in part by the NIHR Biomedical Research Centre of Imperial College NHS Trust. J.B. receives NIHR Senior Investigator Funding. M.N. and J.B. are supported by the Wellcome Trust (207511/Z/17/Z and 203268/Z/16/Z). M.N., J.B. and G.P. are supported by the NIHR University College London Hospitals Biomedical Research Centre. P. Simmonds is supported by the NIHR (NIHR203338). T.S.J. is grateful for funding from the Brain Tumour Charity, Children with Cancer UK, GOSH Children’s Charity, Olivia Hodson Cancer Fund, Cancer Research UK and the NIHR. DIAMONDS is funded by the European Union (Horizon 2020; grant 848196). PERFORM was funded by the European Union (Horizon 2020; grant 668303).Since its first identification in Scotland, over 1000 cases of unexplained pediatric hepatitis in children have been reported worldwide, including 278 cases in the UK 1. Here we report investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator subjects, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in liver, blood, plasma or stool from 27/28 cases. We found low levels of Adenovirus (HAdV) and Human Herpesvirus 6B (HHV-6B), in 23/31 and 16/23 respectively of the cases tested. In contrast, AAV2 was infrequently detected at low titre in blood or liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T-cells and B-lineage cells. Proteomic comparison of liver tissue from cases and healthy controls, identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and in severe cases HHV-6B, may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children.Publisher PDFPeer reviewe

Genomic investigations of unexplained acute hepatitis in children.

Since its first identification in Scotland, over 1000 cases of unexplained pediatric hepatitis in children have been reported worldwide, including 278 cases in the UK 1. Here we report investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator subjects, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in liver, blood, plasma or stool from 27/28 cases. We found low levels of Adenovirus (HAdV) and Human Herpesvirus 6B (HHV-6B), in 23/31 and 16/23 respectively of the cases tested. In contrast, AAV2 was infrequently detected at low titre in blood or liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T-cells and B-lineage cells. Proteomic comparison of liver tissue from cases and healthy controls, identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and in severe cases HHV-6B, may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children