Abstract
Cirrhosis is usually a late-onset and life-threatening disease characterized by fibrotic scarring and inflammation that disrupts liver architecture and function. While it is typically the result of alcoholism or hepatitis viral infection in adults, its etiology in infants is much less understood. In this study, we report 14 children from ten unrelated families presenting with a syndromic form of pediatric liver cirrhosis. By genome/exome sequencing, we found recessive variants in FOCAD segregating with the disease. Zebrafish lacking focad phenocopied the human disease, revealing a signature of altered messenger RNA (mRNA) degradation processes in the liver. Using patient’s primary cells and CRISPR-Cas9-mediated inactivation in human hepatic cell lines, we found that FOCAD deficiency compromises the SKI mRNA surveillance pathway by reducing the levels of the RNA helicase SKIC2 and its cofactor SKIC3. FOCAD knockout hepatocytes exhibited lowered albumin expression and signs of persistent injury accompanied by CCL2 overproduction. Our results reveal the importance of FOCAD in maintaining liver homeostasis and disclose a possible therapeutic intervention point via inhibition of the CCL2/CCR2 signaling axis.
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Data availability
RNA-seq data and processed results have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus public repository under accession number GSE168961. Source data are provided with this paper.
Code availability
No custom code was used for data analysis. All software and packages used are listed in the Methods section.
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Acknowledgements
We are grateful to the families of affected individuals for their participation and kind cooperation in this study. We are also grateful to all members of the Reversade laboratory for constructive discussions and suggestions. We thank R. Weber (Hannover Medical School, Germany) for providing the FOCAD expression construct used for our transient transfections, as well as A. van Hoof (University of Texas Health Science Center at Houston, Texas, USA) and J. Mendell (University of Texas Southwestern Medical Center, Texas, USA) for their insights on the biology of the SKI complex. M.M. was supported by a Career Development Award (CDF Project NR C210812055) from the A*STAR, Biomedical Research Council (Singapore). K.M.G. was supported by the Clinical Research Center grant (IA/CRC/20/1/600002) from India Alliance (India). The authors also thank the King Fahad Medical City Research Center (Saudi Arabia) for the partial support to E.A.F. (grant no. 019-052). B.R. is a fellow of the Branco Weiss Foundation (Switzerland) and the National Research Foundation (Singapore), and an A*STAR and EMBO Young Investigator. This work was also supported by an inaugural Use-Inspired Basic Research (UIBR) central fund and a Brain-Body Initiative (BBI) grant in Neurometabolism to B.R. from the Agency for Science, Technology and Research (A*STAR) in Singapore.
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B.R., K.M.G., M.V. and B.C. initiated the study. B.R. and R.M.T. designed the study. B.R. and P.-M.W. supervised the study. B.R., P.B., A.M.B.-A., V.K., N.O.-H., S.K., M.A. and H.K. coordinated the clinical part of the study. K.M.G., M.V., B.C., A.L., F.A.M., N.A.A.-S., E.A.F., H.A.C., J.D., S.B., B.I., H.P., M.J., K.A.K., S.A.N., E.P.W., M.W., G.S.B., M.D., R.S., E.C., L.R., G.P., H.P.L., B.A., R.M.B. and A.N.A.F. conducted the clinical and genetic evaluation of the patients, the collection of human biological samples and the trio whole genome/exome sequencing for the corresponding affected family. R.M.T. designed and performed all the biochemical, cell culture and in vivo experiments, with help from B.R., T.S.T., P.-M.W., M.M., K.L., N.A.A. and C.Y.C. D.Y.L. and Y.W. conducted the lipidomics analysis on zebrafish livers. G.T. and P.S.L. conducted the clinical curation of the FOCAD variants. B.W. performed the structural analysis of the missense FOCAD variants. R.M.T. conducted all the rest of the data processing and analysis. B.R. and R.M.T. wrote the manuscript with input from all co-authors and performed all revisions.
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P.B., A.M.B.-A., V.K., N.O.-H. and S.K. are employed by and receive a salary from Centogene AG (exome sequencing is among the commercially available tests). K.M.G. is founder and director of Suma Genomics (exome sequencing is among the commercially available tests). The remaining authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Clinical data from patient F2-II:2 (France).
a, Photograph of the patient showing a triangular face, high forehead and plagiocephaly. b, Liver biopsy at 2 years and 4 months. Periodic acid Schiff staining (PAS, top left) showed elevated intrahepatocellular glycogen. Gordon and Sweet’s silver staining (bottom left) revealed some regions of the liver parenchyma with an abnormal reticular fiber pattern (yellow arrows). H&E staining (top middle and right) showed presence of fibrogenic connective tissue (black frame) and lipid vesicles in hepatocytes indicative of steatosis (green arrows). Picrosirius red staining (bottom middle and right) confirmed the presence of fibrotic bands (black frame) and highlighted the presence of pericellular collagen fibers among hepatocytes (white arrows). Scale bars, 50 μm (bottom left) and 25 μm (bottom right).
Extended Data Fig. 2 focad is maternally contributed in zebrafish.
a, Schematic diagram of the genomic structure of focad in zebrafish. Two germline deletion mutations were selected to generate focad knockouts using CRISPR-Cas9 technology. The focad gene comprises 44 exons (bars). The blue line highlights the region targeted by the CRISPR guide RNA (gRNA) in exon 4. Black arrows point where the genomic deletions of 7 (focadΔ7) and 8 (focadΔ8) base pairs have occurred, which result in out-of-frame alleles creating early stop codons. b, Temporal RT-qPCR quantification of the expression levels of focad relative to β-actin during the 7 days postfertilization. focad is maternally contributed to the egg. Data are presented as mean ± s.e.m. (n = 2 biological replicates of 30 to 50 embryos).
Extended Data Fig. 3 Overlap of the main clinical manifestations seen in FOCAD-deficient patients and THES type 1 and type 2.
Data related to both types of THES were extracted from Bourgeois et al.57.
Supplementary information
Supplementary Information
Supplementary Tables 6–8 and Note.
Supplementary Tables
Supplementary Tables 1–5.
Source data
Source Data Fig. 3
Unprocessed western blots.
Source Data Fig. 5
Unprocessed western blots.
Source Data Fig. 6
Unprocessed western blots.
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Moreno Traspas, R., Teoh, T.S., Wong, PM. et al. Loss of FOCAD, operating via the SKI messenger RNA surveillance pathway, causes a pediatric syndrome with liver cirrhosis. Nat Genet 54, 1214–1226 (2022). https://doi.org/10.1038/s41588-022-01120-0
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DOI: https://doi.org/10.1038/s41588-022-01120-0
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