Two Novel Pathogenic Variants of TJP2 Gene and the Underlying Molecular Mechanisms in Progressive Familial Intrahepatic Cholestasis Type 4 Patients

Abstract

Progressive familial intrahepatic cholestasis (PFIC) is an autosomal recessive inherited disease that accounts for 10%–15% childhood cholestasis and could lead to infant disability or death. There are three well-established types of PFIC (1–3), caused by mutations in the ATP8B1, ABCB11, and ABCB4 genes. Biallelic pathogenic variants in the tight junction protein 2 gene (TJP2) were newly reported as a cause for PFIC type 4; however, only a limited number of patients and undisputable variants have been reported for TJP2, and the underlying mechanism for PFIC 4 remains poorly understood. To explore the diagnostic yield of TJP2 analysis in suspected PFIC patients negative for the PFIC1–3 mutation, we designed a multiplex polymerase chain reaction-based next-generation sequencing method to analyze TJP2 gene variants in 267 PFIC patients and identified biallelic rare variants in three patients, including three known pathogenic variants and two novel variants in three patients. By using CRISPR-cas9 technology, we demonstrated that TJP2 c.1202A > G was pathogenic at least partially by increasing the expression and nuclear localization of TJP2 protein. With the minigene assay, we showed that TJP2 c.2668-11A > G was a new pathogenic variant by inducing abnormal splicing of TJP2 gene and translation of prematurely truncated TJP2 protein. Furthermore, knockdown of TJP2 protein by siRNA technology led to inhibition of cell proliferation, induction of apoptosis, dispersed F-actin, and disordered microfilaments in LO2 and HepG2celles. Global gene expression profiling of TJP2 knockdown LO2 cells and HepG2 cells identified the dysregulated genes involved in the regulation of actin cytoskeleton. Microtubule cytoskeleton genes were significantly downregulated in TJP2 knockdown cells. The results of this study demonstrate that TJP2 c.1202A > G and TJP2 c.2668-11A > G are two novel pathogenic variants and the cytoskeleton-related functions and pathways might be potential molecular pathogenesis for PFIC.

New Set of Isobaric Labeling Reagents for Quantitative 16Plex Proteomics

“…Basically, these differential proteins of MCF7 responding to culturing in a glucose-rich medium are mainly involved in the two functions, membrane-related such as DNAJC1, NDUFA2, TJP2, TNPO2, and PLEKHF2 and nucleus-located such as HDAC1 and TARDBP, and are assumed to be involved in the formation of protein complexes. Their dynamic behaviors of protein abundance during the 4 h culturing period presented in Figure E, however, exhibited quite diverse modes.…”

A Rare Case of Progressive Familial Intrahepatic Cholestasis Type 4: A Case Report and Literature Review

“…Based on clinical presentation, liver biopsies, and molecular testing, our patient was diagnosed with PFIC 4. Few reported cases were available in the literature [5,[7][8][9][10][11]. The clinical manifestations of PFIC 4 in the neonatal period range from mild neonatal cholestasis and obstructive jaundice [4] to a more severe clinical course [4,5,7].…”

“…There are few case reports of PFIC 4 to date [5,[7][8][9][10][11]. We report the first PFIC 4 case in the Middle East involving a 46-day-old male infant with chronic cholestatic liver disease and low gammaglutamyltransferase (GGT) diagnosed with TJP2-related PFIC 4 and treated with a liver transplant.…”

Genetic alterations and molecular mechanisms underlying hereditary intrahepatic cholestasis

“…Additionally, there were dilated tubules, reduced microvilli density, and decreased expression of the tubule membrane transporter BSEP ( Xu et al, 2021 ). In vitro experiments have shown that inhibiting TJP2 expression using siRNA can inhibit the proliferation of L02 and HepG2 cells, induce cell apoptosis, and cause microfilament disturbance ( Tang et al, 2021 ).…”