Human iPSC-derived hepatocyte system models cholestasis with tight junction protein 2 deficiency.

Background & Aims: The truncating mutations in tight junction protein 2 (TJP2) cause progressive cholestasis, liver failure, and hepatocyte carcinogenesis. Due to the lack of effective model systems, there are no targeted medications for the liver pathology with TJP2 deficiency. We leveraged the technologies of patient-specific induced pluripotent stem cells (iPSC) and CRISPR genome-editing, and we aim to establish a disease model which recapitulates phenotypes of patients with TJP2 deficiency. Methods: We differentiated iPSC to hepatocyte-like cells (iHep) on the Transwell membrane in a polarized monolayer. Immunofluorescent staining of polarity markers was detected by a confocal microscope. The epithelial barrier function and bile acid transport of bile canaliculi were quantified between the two chambers of Transwell. The morphology of bile canaliculi was measured in iHep cultured in the Matrigel sandwich system using a fluorescent probe and live-confocal imaging. Results: The iHep differentiated from iPSC with TJP2 mutations exhibited intracellular inclusions of disrupted apical membrane structures, distorted canalicular networks, altered distribution of apical and basolateral markers/transporters. The directional bile acid transport of bile canaliculi was compromised in the mutant hepatocytes, resembling the disease phenotypes observed in the liver of patients. Conclusions: Our iPSC-derived in vitro hepatocyte system revealed canalicular membrane disruption in TJP2 deficient hepatocytes and demonstrated the ability to model cholestatic disease with TJP2 deficiency to serve as a platform for further pathophysiologic study and drug discovery. Lay summary: We investigated a genetic liver disease, progressive familial intrahepatic cholestasis (PFIC), which causes severe liver disease in newborns and infants due to a lack of gene called TJP2. By using cutting-edge stem cell technology and genome editing methods, we established a novel disease modeling system in cell culture experiments. Our experiments demonstrated that the lack of TJP2 induced abnormal cell polarity and disrupted bile acid transport. These findings will lead to the subsequent investigation to further understand disease mechanisms and develop an effective treatment.

Study of Acute Liver Failure in Children Using Next Generation Sequencing Technology.

OBJECTIVE: To use next generation sequencing (NGS) technology to identify undiagnosed, monogenic diseases in a cohort of children who suffered from acute liver failure (ALF) without an identifiable etiology. STUDY DESIGN: We identified 148 under 10 years of age admitted to King's College Hospital, London, with ALF of indeterminate etiology between 2000 and 2018. A custom NGS panel of 64 candidate genes known to cause ALF and/or metabolic liver disease was constructed. Targeted sequencing was carried out on 41 children in whom DNA samples were available. Trio exome sequencing was performed on 4 children admitted during 2019. A comparison of the clinical characteristics of those identified with biallelic variants against those without biallelic variants was then made. RESULTS: Homozygous and compound heterozygous variants were identified in 8 out of 41 children (20%) and 4 out of 4 children (100%) in whom targeted and exome sequencing were carried out, respectively. The genes involved were NBAS (3 children); DLD (2 children); and CPT1A, FAH, LARS1, MPV17, NPC1, POLG, SUCLG1, and TWINK (1 each). The 12 children who were identified with biallelic variants were younger at presentation and more likely to die in comparison with those who did not: median age at presentation of 3 months and 30 months and survival rate 75% and 97%, respectively. CONCLUSIONS: NGS was successful in identifying several specific etiologies of ALF. Variants in NBAS and mitochondrial DNA maintenance genes were the most common findings. In the future, a rapid sequencing NGS workflow could help in reaching a timely diagnosis and facilitate clinical decision making in children with ALF.

Cholestasis Due to USP53 Deficiency.

OBJECTIVES: Although a number of genetic forms of cholestasis have been identified, the genetic etiology of disease remains unidentified in a subset of cholestasis patients. METHODS: Whole exome sequencing (WES) was performed in DNA from patients diagnosed with cholestasis, at different points on the continuum from progressive familial intrahepatic cholestasis to benign recurrent intrahepatic cholestasis, in whom no disease mutations in known cholestasis genes had been identified. Candidate genes were then assessed in a larger patient sample, by targeted next-generation sequencing (NGS). Disease features at presentation and follow-up were collected from available medical records. RESULTS: By WES, we identified 3 patients with homozygous mutations in USP53. Screening of USP53 in a larger set of patients identified 4 additional patients with homozygous mutations in USP53. Six of the 7 patients had deletion mutations, and 1 had a missense mutation; 3 of the patients were siblings, all bearing a deletion that also disrupted neighboring MYOZ2. Age of onset ranged from early infancy to adolescence. Cholestasis tended to be biochemically mild and intermittent, and responsive to medication. Liver fibrosis was, however, present in all 4 patients who were biopsied, and splenomegaly was apparent in 5 of 7 at last ultrasound. CONCLUSIONS: Two groups recently identified patients with liver disease and mutation in USP53. We have now identified biallelic mutation in USP53 in 7 further patients with cholestasis, from 5 families. Most individuals had evidence of chronic liver disease, and long-term follow-up is recommended.

An expanded role for heterozygous mutations of ABCB4, ABCB11, ATP8B1, ABCC2 and TJP2 in intrahepatic cholestasis of pregnancy.

Intrahepatic cholestasis of pregnancy (ICP) affects 1/140 UK pregnancies; with pruritus, hepatic impairment and elevated serum bile acids. Severe disease is complicated by spontaneous preterm delivery and stillbirth. Previous studies have reported mutations in hepatocellular transporters (ABCB4, ABCB11). High throughput sequencing in 147 patients was performed in the transporters ABCB4, ABCB11, ATP8B1, ABCC2 and tight junction protein 2 (TJP2). Twenty-six potentially damaging variants were identified with the following predicted protein changes: Twelve ABCB4 mutations - Arg47Gln, Met113Val, Glu161Gly, Thr175Ala, Glu528Glyfs*6, Arg590Gln, Ala601Ser, Glu884Ter, Gly722Ala, Tyr775Met (x2), Trp854Ter. Four potential ABCB11 mutations - Glu297Gly (x3) and a donor splice site mutation (intron 19). Five potential ATP8B1 mutations - Asn45Thr (x3), and two others, Glu114Gln and Lys203Glu. Two ABCC2 mutations - Glu1352Ala and a duplication (exons 24 and 25). Three potential mutations were identified in TJP2; Thr62Met (x2) and Thr626Ser. No patient harboured more than one mutation. All were heterozygous. An additional 545 cases were screened for the potential recurrent mutations of ATP8B1 (Asn45Thr) and TJP2 (Thr62Met) identifying three further occurrences of Asn45Thr. This study has expanded known mutations in ABCB4 and ABCB11 and identified roles in ICP for mutations in ATP8B1 and ABCC2. Possible novel mutations in TJP2 were also discovered.

Mutations in DCDC2 (doublecortin domain containing protein 2) in neonatal sclerosing cholangitis.

BACKGROUND & AIMS: Neonatal sclerosing cholangitis (NSC) is a severe neonatal-onset cholangiopathy commonly leading to liver transplantation (LT) for end-stage liver disease in childhood. Liver biopsy findings histopathologically resemble those in biliary atresia (BA); however, in NSC extrahepatic bile ducts are patent, whilst in BA their lumina are obliterated. NSC is commonly seen in consanguineous kindreds, suggesting autosomal recessive inheritance. METHODS: From 29 NSC patients (24 families) identified, DNA was available in 24 (21 families). Thirteen (7 male) patients (12 families) of consanguineous parentage were selected for whole exome sequencing. Sequence variants were filtered for homozygosity, pathogenicity, minor allele frequency, quality score, and encoded protein expression pattern. RESULTS: Four of 13 patients were homozygous and two were compound heterozygous for mutations in the doublecortin domain containing 2 gene (DCDC2), which encodes DCDC2 protein and is expressed in cholangiocyte cilia. Another 11 patients were sequenced: one (with one sibling pair) was compound heterozygous for DCDC2 mutations. All mutations were protein-truncating. In available liver tissue from patients with DCDC2 mutations, immunostaining for human DCDC2 and the ciliary protein acetylated alpha-tubulin (ACALT) showed no expression (n=6) and transmission electron microscopy found that cholangiocytes lacked primary cilia (n=5). DCDC2 and ACALT were expressed in NSC patients without DCDC2 mutations (n=22). Of the patients carrying DCDC2 mutations, one died awaiting LT; five came to LT, of whom one died 2years later. The other 4 are well. CONCLUSION: Among 24 NSC patients with available DNA, 7 had mutations in DCDC2 (6 of 19 families). NSC patients in substantial proportion harbour mutations in DCDC2. Their disease represents a novel liver-based ciliopathy. LAY SUMMARY: Neonatal sclerosing cholangitis (NSC) is a rare genetic form of liver disease presenting in infancy. Through next generation sequencing we identified mutations in the gene encoding for doublecortin domain containing 2 (DCDC2) protein in a group of NSC children. DCDC2 is a signalling and structural protein found in primary cilia of cholangiocytes. Cholangiocytes are the cells forming the biliary system which is the draining system of the liver.

Mutations in TJP2, encoding zona occludens 2, and liver disease.

Progressive familial intrahepatic cholestasis is a clinical description of a phenotype, which we now realize has several different genetic aetiologies. The identification of the underlying genetic defects has helped to elucidate important aspects of liver physiology. The latest addition to this family of diseases is tight junction protein 2 (TJP2) deficiency. This protein is also known as zona occludens 2 (ZO-2). The patients, so far presented, all have homozygous, protein-truncating mutations. A complete absence of this protein was demonstrated. These children presented with severe liver disease, some manifesting extrahepatic features. By contrast, embryonic-lethality was seen in ZO-2 knockout mice. This discovery highlights important differences, not just between species, but also between different epithelia in humans. This commentary discusses the recently presented findings, and some of the issues that arise.

Mutations in TJP2 cause progressive cholestatic liver disease.

Elucidating genetic causes of cholestasis has proved to be important in understanding the physiology and pathophysiology of the liver. Here we show that protein-truncating mutations in the tight junction protein 2 gene (TJP2) cause failure of protein localization and disruption of tight-junction structure, leading to severe cholestatic liver disease. These findings contrast with those in the embryonic-lethal knockout mouse, highlighting differences in redundancy in junctional complexes between organs and species.