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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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.
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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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).
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.
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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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