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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Elpek, G. Ö. Cellular and molecular mechanisms in the pathogenesis of liver fibrosis: an update. World J. Gastroenterol. 20, 7260–7276 (2014).
Seki, E. & Brenner, D. A. Recent advancement of molecular mechanisms of liver fibrosis. J. Hepatobiliary Pancreat. Sci. 22, 512–518 (2015).
Friedman, S. L. Hepatic fibrosis—overview. Toxicology 254, 120–129 (2008).
Ding, B.-S. et al. Divergent angiocrine signals from vascular niche balance liver regeneration and fibrosis. Nature 505, 97–102 (2014).
Mederacke, I. et al. Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology. Nat. Commun. 4, 2823 (2013).
Wynn, T. A. & Ramalingam, T. R. Mechanisms of fibrosis: therapeutic translation for fibrotic disease. Nat. Med. 18, 1028–1040 (2012).
Schuppan, D. & Afdhal, N. H. Liver cirrhosis. Lancet 371, 838–851 (2008).
Mokdad, A. A. et al. Liver cirrhosis mortality in 187 countries between 1980 and 2010: a systematic analysis. BMC Med. 12, 145 (2014).
Asrani, S. K., Devarbhavi, H., Eaton, J. & Kamath, P. S. Burden of liver diseases in the world. J. Hepatol. 70, 151–171 (2019).
Yoshiji, H. et al. Evidence-based clinical practice guidelines for liver cirrhosis 2020. Hepatol. Res. 51, 725–749 (2021).
Brockschmidt, A. et al. KIAA1797/FOCAD encodes a novel focal adhesion protein with tumour suppressor function in gliomas. Brain 135, 1027–1041 (2012).
Brand, F. et al. FOCAD loss impacts microtubule assembly, G2/M progression and patient survival in astrocytic gliomas. Acta Neuropathol. 139, 175–192 (2020).
Weren, R. D. A. et al. Germline deletions in the tumour suppressor gene FOCAD are associated with polyposis and colorectal cancer development. J. Pathol. 236, 155–164 (2015).
Wiech, T. et al. Genome-wide analysis of genetic alterations in Barrett’s adenocarcinoma using single nucleotide polymorphism arrays. Lab. Invest. 89, 385–397 (2009).
Toh-E, A., Guerry, P. & Wickner, R. B. Chromosomal superkiller mutants of Saccharomyces cerevisiae. J. Bacteriol. 136, 1002–1007 (1978).
Brown, J. T., Bai, X. & Johnson, A. W. The yeast antiviral proteins Ski2p, Ski3p, and Ski8p exist as a complex in vivo. RNA 6, 449–457 (2000).
Widner, W. R. & Wickner, R. B. Evidence that the SKI antiviral system of Saccharomyces cerevisiae acts by blocking expression of viral mRNA. Mol. Cell. Biol. 13, 4331–4341 (1993).
Synowsky, S. A. & Heck, A. J. R. The yeast Ski complex is a hetero-tetramer. Protein Sci. 17, 119–125 (2008).
Halbach, F., Reichelt, P., Rode, M. & Conti, E. The yeast Ski complex: crystal structure and RNA channeling to the exosome complex. Cell 154, 814–826 (2013).
Anderson, J. S. & Parker, R. P. The 3′ to 5′ degradation of yeast mRNAs is a general mechanism for mRNA turnover that requires the SKI2 DEVH box protein and 3′ to 5′ exonucleases of the exosome complex. EMBO J. 17, 1497–1506 (1998).
Tuck, A. C. et al. Mammalian RNA decay pathways are highly specialized and widely linked to translation. Molecular Cell 77, 1222–1236.e13 (2020).
Lee, W. S. & Sokol, R. J. Mitochondrial hepatopathies: advances in genetics and pathogenesis. Hepatology 45, 1555–1565 (2007).
Fabris, L. et al. Pathobiology of inherited biliary diseases: a roadmap to understand acquired liver diseases. Nat. Rev. Gastroenterol. Hepatol. 16, 497–511 (2019).
Ozen, H. Glycogen storage diseases: new perspectives. World J. Gastroenterol. 13, 2541–2553 (2007).
Lange, H. et al. RST1 and RIPR connect the cytosolic RNA exosome to the Ski complex in Arabidopsis. Nat. Commun. 10, 3871 (2019).
Desmet, F.-O. et al. Human Splicing Finder: an online bioinformatics tool to predict splicing signals. Nucleic Acids Res. 37, e67 (2009).
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).
Schwabe, R. F. & Maher, J. J. Lipids in liver disease: looking beyond steatosis. Gastroenterology 142, 8–11 (2012).
Ding, H., Wang, J., Ren, H. & Shi, X. Lipometabolism and glycometabolism in liver diseases. BioMed Res. Int. 2018, 1287127 (2018).
Uhlar, C. M. & Whitehead, A. S. Serum amyloid A, the major vertebrate acute-phase reactant. Eur. J. Biochem. 265, 501–523 (1999).
Aly, H. H. et al. RNA exosome complex regulates stability of the hepatitis B virus X-mRNA transcript in a non-stop-mediated (NSD) RNA quality control mechanism. J. Biol. Chem. 291, 15958–15974 (2016).
Zhu, B. et al. The human PAF complex coordinates transcription with events downstream of RNA synthesis. Genes Dev. 19, 1668–1673 (2005).
Qu, X. et al. The human DEVH-box protein Ski2w from the HLA is localized in nucleoli and ribosomes. Nucleic Acids Res. 26, 4068–4077 (1998).
Xu, L. et al. Human hepatic stellate cell lines, LX-1 and LX-2: new tools for analysis of hepatic fibrosis. Gut 54, 142–151 (2005).
Waki, K. et al. Establishment of functional telomerase immortalized human hepatocytes and a hepatic stellate cell line for telomere-targeting anticancer drug development. Cancer Sci. 101, 1678–1685 (2010).
Border, W. A. & Noble, N. A. Transforming growth factor beta in tissue fibrosis. N. Engl. J. Med. 331, 1286–1292 (1994).
Junge, N., Sharma, A. D. & Ott, M. About cytokeratin 19 and the drivers of liver regeneration. J. Hepatol. 68, 5–7 (2018).
Yoneda, N. et al. Epidermal growth factor induces cytokeratin 19 expression accompanied by increased growth abilities in human hepatocellular carcinoma. Lab. Invest. 91, 262–272 (2011).
Chen, J. et al. Hepatic lipocalin 2 promotes liver fibrosis and portal hypertension. Sci. Rep. 10, 15558 (2020).
Xu, Y. et al. Lipocalin-2 protects against diet-induced nonalcoholic fatty liver disease by targeting hepatocytes. Hepatol. Commun. 3, 763–775 (2019).
Mungrue, I. N., Pagnon, J., Kohannim, O., Gargalovic, P. S. & Lusis, A. J. CHAC1/MGC4504 is a novel proapoptotic component of the unfolded protein response, downstream of the ATF4-ATF3-CHOP cascade. J. Immunol. 182, 466–476 (2009).
Magne, L. et al. ATF4 and the integrated stress response are induced by ethanol and cytochrome P450 2E1 in human hepatocytes. J. Hepatol. 54, 729–737 (2011).
Henriksen, J. H. et al. Dynamics of albumin in plasma and ascitic fluid in patients with cirrhosis. J. Hepatol. 34, 53–60 (2001).
Feldmann, G., Penaud-Laurencin, J., Crassous, J. & Benhamou, J. P. Albumin synthesis by human liver cells: its morphological demonstration. Gastroenterology 63, 1036–1048 (1972).
Boon, R. et al. Amino acid levels determine metabolism and CYP450 function of hepatocytes and hepatoma cell lines. Nat. Commun. 11, 1393 (2020).
Villeneuve, J.-P. & Pichette, V. Cytochrome P450 and liver diseases. Curr. Drug Metab. 5, 273–282 (2004).
Siegel, R. C., Chen, K. H., Greenspan, J. S. & Aguiar, J. M. Biochemical and immunochemical study of lysyl oxidase in experimental hepatic fibrosis in the rat. Proc. Natl Acad. Sci. USA 75, 2945–2949 (1978).
Vadasz, Z. et al. Abnormal deposition of collagen around hepatocytes in Wilson’s disease is associated with hepatocyte specific expression of lysyl oxidase and lysyl oxidase like protein-2. J. Hepatol. 43, 499–507 (2005).
Mandrekar, P., Ambade, A., Lim, A., Szabo, G. & Catalano, D. An essential role for monocyte chemoattractant protein-1 in alcoholic liver injury: regulation of proinflammatory cytokines and hepatic steatosis in mice. Hepatology 54, 2185–2197 (2011).
Kobayashi, K. et al. Role of monocyte chemoattractant protein-1 in liver fibrosis with transient myeloproliferative disorder in down syndrome. Hepatol. Commun. 2, 230–236 (2018).
Xie, J. et al. Macrophage migration inhibitor factor upregulates MCP-1 expression in an autocrine manner in hepatocytes during acute mouse liver injury. Sci. Rep. 6, 27665 (2016).
Li, S. et al. TSLP protects against liver I/R injury via activation of the PI3K/Akt pathway. JCI Insight 4, e129013 (2019).
Sawa, Y. et al. Hepatic interleukin-7 expression regulates T cell responses. Immunity 30, 447–457 (2009).
Affò, S. et al. CCL20 mediates lipopolysaccharide induced liver injury and is a potential driver of inflammation and fibrosis in alcoholic hepatitis. Gut 63, 1782–1792 (2014).
Abul-Husn, N. S. et al. A protein-truncating HSD17B13 variant and protection from chronic liver disease. N. Engl. J. Med. 378, 1096–1106 (2018).
Esmaili, S. et al. Core liver homeostatic co-expression networks are preserved but respond to perturbations in an organism- and disease-specific manner. Cell Systems 12, 432–445.e7 (2021).
Bourgeois, P. et al. Tricho-hepato-enteric syndrome mutation update: mutations spectrum of TTC37 and SKIV2L, clinical analysis and future prospects. Hum. Mutat. 39, 774–789 (2018).
Hartley, J. L. et al. Mutations in TTC37 cause trichohepatoenteric syndrome (phenotypic diarrhea of infancy). Gastroenterology 138, 2388–2398 (2010). 2398.e1–2.
Hiejima, E. et al. Tricho-hepato-enteric syndrome with novel SKIV2L gene mutations: a case report. Medicine (Baltimore) 96, e8601 (2017).
Fabre, A. et al. SKIV2L mutations cause syndromic diarrhea, or trichohepatoenteric syndrome. Am. J. Hum. Genet. 90, 689–692 (2012).
Sinha, N. K. et al. EDF1 coordinates cellular responses to ribosome collisions. eLife 9, e58828 (2020).
Wu, C. C.-C., Peterson, A., Zinshteyn, B., Regot, S. & Green, R. Ribosome collisions trigger general stress responses to regulate cell fate. Cell 182, 404–416.e14 (2020).
Queck, A. et al. Systemic MCP-1 levels derive mainly from injured liver and are associated with complications in cirrhosis. Front. Immunol. 11, 354 (2020).
Ambade, A. et al. Pharmacological inhibition of CCR2/5 signaling prevents and reverses alcohol-induced liver damage, steatosis, and inflammation in mice. Hepatology 69, 1105–1121 (2019).
Baeck, C. et al. Pharmacological inhibition of the chemokine CCL2 (MCP-1) diminishes liver macrophage infiltration and steatohepatitis in chronic hepatic injury. Gut 61, 416–426 (2012).
Teng, K.-Y. et al. Blocking the CCL2-CCR2 axis using CCL2 neutralizing antibody is an effective therapy for hepatocellular cancer in a mouse model. Mol. Cancer Ther. 16, 312–322 (2017).
Anstee, Q. M. et al. Cenicriviroc for the treatment of liver fibrosis in adults with nonalcoholic steatohepatitis: AURORA Phase 3 study design. Contemp. Clin. Trials 89, 105922 (2020).
Clark, M. M. et al. Diagnosis of genetic diseases in seriously ill children by rapid whole-genome sequencing and automated phenotyping and interpretation. Sci. Transl. Med. 11, eaat6177 (2019).
Kumar, P., Henikoff, S. & Ng, P. C. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat. Protoc. 4, 1073–1081 (2009).
Adzhubei, I. A. et al. A method and server for predicting damaging missense mutations. Nat. Methods 7, 248–249 (2010).
Schwarz, J. M., Rödelsperger, C., Schuelke, M. & Seelow, D. MutationTaster evaluates disease-causing potential of sequence alterations. Nat. Methods 7, 575–576 (2010).
Rentzsch, P., Witten, D., Cooper, G. M., Shendure, J. & Kircher, M. CADD: predicting the deleteriousness of variants throughout the human genome. Nucleic Acids Res. 47, D886–D894 (2019).
Quang, D., Chen, Y. & Xie, X. DANN: a deep learning approach for annotating the pathogenicity of genetic variants. Bioinformatics 31, 761–763 (2015).
Shamsani, J. et al. A plugin for the ensembl variant effect predictor that uses MaxEntScan to predict variant spliceogenicity. Bioinformatics 35, 2315–2317 (2019).
Pertea, M., Lin, X. & Salzberg, S. L. GeneSplicer: a new computational method for splice site prediction. Nucleic Acids Res. 29, 1185–1190 (2001).
Richards, S. et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet. Med. 17, 405–424 (2015).
Vangipuram, M., Ting, D., Kim, S., Diaz, R. & Schüle, B. Skin punch biopsy explant culture for derivation of primary human fibroblasts. J. Vis. Exp. 77, e3779 (2013).
Tang, W. J. Blot-affinity purification of antibodies. Methods Cell. Biol. 37, 95–104 (1993).
Jao, L.-E., Wente, S. R. & Chen, W. Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc. Natl Acad. Sci. USA 110, 13904–13909 (2013).
Bligh, E. G. & Dyer, W. J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917 (1959).
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
Nature Reviews Gastroenterology & Hepatology (2022)