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

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

Abstract 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 DCDC2, encoding doublecortin domain containing 2 (DCDC2), 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 DCDC2, one patient died awaiting LT; five came to LT, of whom one died 2 years 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

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

Mutations in TJP2 cause progressive cholestatic liver disease

The elucidation of genetic causes of cholestasis has proved to be important in understanding the physiology and pathophysiology of the liver. Protein-truncating mutations in the tight junction protein 2 gene (TJP2) are shown to cause failure of protein localisation, with disruption of tight-junction structure leading to severe cholestatic liver disease. This contrasts with the embryonic-lethal knockout mouse, highlighting differences in redundancy in junctional complexes between organs and species