SUGP2 p.(Arg639Gln) variant is involved in the pathogenesis of hemochromatosis via the CIRBP/BMPER signaling pathway.

Pathogenic variants in HFE and non-HFE genes have been identified in hemochromatosis in different patient populations, but there are still a certain number of patients with unexplained primary iron overload. We recently identified in Chinese patients a recurrent p.(Arg639Gln) variant in SURP and G-patch domain containing 2 (SUGP2), a potential mRNA splicing-related factor. However, the target gene of SUGP2 and affected iron-regulating pathway remains unknown. We aimed to investigate the pathogenicity and underlying mechanism of this variant in hemochromatosis. RNA-seq analysis revealed that SUGP2 knockdown caused abnormal alternative splicing of CIRBP pre-mRNA, resulting in an increased normal splicing form of CIRBP V1, which in turn increased the expression of BMPER by enhancing its mRNA stability and translation. Furthermore, RNA-protein pull-down and RNA immunoprecipitation assays revealed that SUGP2 inhibited splicing of CIRBP pre-mRNA by a splice site variant at CIRBP c.492 and was more susceptible to CIRBP c.492 C/C genotype. Cells transfected with SUGP2 p.(Arg639Gln) vector showed up-regulation of CIRBP V1 and BMPER expression and down-regulation of pSMAD1/5 and HAMP expression. CRISPR-Cas9 mediated SUGP2 p.(Arg622Gln) knock-in mice showed increased iron accumulation in the liver, higher total serum iron, and decreased serum hepcidin level. A total of 10 of 54 patients with hemochromatosis (18.5%) harbored the SUGP2 p.(Arg639Gln) variant and carried CIRBP c.492 C/C genotype, and had increased BMPER expression in the liver. Altogether, the SUGP2 p.(Arg639Gln) variant down-regulates hepcidin expression through the SUGP2/CIRBP/BMPER axis, which may represent a novel pathogenic factor for hemochromatosis.

Laboratory and clinical evaluation of a microarray for the detection of ATP7B mutations in Wilson disease in China.

BACKGROUND AND OBJECTIVE: Wilson disease (WD) is an autosomal recessive copper metabolic disorder caused by mutations in ATP7B. Sanger sequencing is currently used for ATP7B variant identification. However, the ATP7B gene contains 21 exons, which makes sequencing of the entire gene both complex and time-consuming. Therefore, a simpler assay is urgently needed. METHODS: We performed a laboratory and clinical evaluation of an oligonucleotide microarray for the detection of 24 ATP7B recurrent mutations (except p.P992L) in Chinese patients with WD. RESULTS: The accuracy of the microarray was evaluated by screening for ATP7B mutations in 126 patients including 106 suspected WD samples and 20 patients with other liver diseases as negative control. Results were confirmed by Sanger sequencing. We established a reliable microarray system for the rapid detection of the 24 ATP7B mutations, with a sensitivity of 30 ng/test genomic DNA and specificity of 100% for all loci; the coefficient of variation in repeatability tests was <10%. Clinical evaluation showed an overall concordance between the microarray detection and sequencing of 100%, and 81.13% (86/106) of suspected WD cases showed ATP7B mutations by microarray detection. Microarray and Sanger sequencing identified p.R778L (50.94%), p.A874V (17.92%), p.P992L (11.32%), p.V1106I (11.32%), and p.I1148T (6.60%) as the most common mutations in WD patients. CONCLUSIONS: Our microarray system is customizable and easily used for high-throughput detection of certain recurrent ATP7B mutations, providing a simpler method suitable for WD genetic diagnosis in China.

14-kDa phosphohistidine phosphatase is a potential therapeutic target for liver fibrosis.

Liver fibrosis, a major cause of morbidity and mortality worldwide, leads to liver damage, seriously threatening human health. In our previous study, we demonstrated that 14 kDa phosphohistidine phosphatase (PHP14) was upregulated in fibrotic liver tissue and involved in the migration and lamellipodia formation of hepatic stellate cells (HSCs). In this study, we evaluated PHP14 as a therapeutic target for liver fibrosis and investigated the mechanism by which it mediates liver fibrosis. AAV-shPhpt1 administration significantly attenuates CCl4-induced liver fibrosis in mice. In particular, fibrosis-associated inflammatory infiltration was significantly suppressed after PHP14 knockdown. Mechanistically, PHP14 regulated macrophage recruitment, infiltration, and migration by affecting podosome formation of macrophages. Inhibition of PHP14 decreased the expression of the fibrogenic signature at the early stage of liver fibrogenesis and the activation of HSCs in vivo. Thus, PHP14 can be considered a potential therapeutic target for liver fibrosis.NEW & NOTEWORTHY PHP14 inhibition via adeno-associated virus (AAV)-mediated gene silencing could potently attenuate carbon tetrachloride (CCl4)-induced liver fibrosis. PHP14 could regulate the migration of macrophages to the site of injury in vivo. PHP14 knockdown in vivo influenced the environment of fibrogenesis and relevant signaling pathways, subsequently affecting myofibroblast activation.

Complex ATP7B mutation patterns in Wilson disease and evaluation of a yeast model for functional analysis of variants.

Wilson disease (WD) is a rare autosomal recessive genetic disorder that is associated with various mutations in the ATP7B gene. Although ATP7B variants are frequently identified, the exact mutation patterns remain unknown because of the absence of pedigree studies, and the functional consequences of individual ATP7B variants remain to be clarified. In this study, we recruited 65 clinically diagnosed WD patients from 60 unrelated families. Pedigree analysis showed that besides several ATP7B homozygous variants (8/65, 12.3%), compound heterozygous variants (43/65, 66.2%) were present in the majority of WD patients. There were 20% of the patients had one (12/65, 18.5%) or multiple (1/65, 1.5%) variants in only a single allele, characterized by a high ratio of splicing or frameshift variants. Nine ATP7B variants were cloned into the pAG426GPD yeast expression vector to evaluate their functional consequences, and the results suggested different degrees of functional disruption from mild or uncertain to severe, consistent with the corresponding phenotypes. Our study revealed the complex ATP7B mutation patterns in WD patients and the applicability of a yeast model system to the evaluation of the functional consequences of ATP7B variants, which is essential for WD cases that are difficult to interpret.

A novel SLC40A1 p.Y333H mutation with gain of function of ferroportin: A recurrent cause of haemochromatosis in China.

BACKGROUND & AIMS: Haemochromatosis type 4, also known as ferroportin disease, is an autosomal dominant genetic disorder caused by pathogenic mutations in the SLC40A1 gene, which encodes ferroportin 1 (FPN1). We have identified a novel SLC40A1 p.Y333H mutation in our previous study. In the present study, we tried to investigate the frequency and pathogenicity of the SLC40A1 p.Y333H mutation in haemochromatosis in China. METHODS: Patients were analysed for SLC40A1 p.Y333H as well as mutations in the other classic haemochromatosis-related genes by Sanger sequencing. To analyse iron export capacity of the SLC40A1 p.Y333H mutant, the 293T cells were transfected with the SLC40A1 p.Y333H construct and then treated with hepcidin after exposure to ferric ammonium citrate. Cellular localization of mutant FPN1, expression of FPN1 and intracellular ferritin were analysed by immunofluorescence and Western blotting. RESULTS: Of 22 unrelated cases with primary iron overload, three cases (3/22, 13.6%) harboured the SLC40A1 p.Y333H, with no missense mutations identified in any other classical haemochromatosis-related genes including HFE, HJV, HAMP and TFR2. Pedigree analysis showed that three probands and the son of one proband had haemochromatosis of stage 3, while the son of another proband with age of 16 showed elevated transferrin saturation but normal serum ferritin level. In vitro studies showed the mutant p.Y333H ferroportin was resistant to hepcidin, affecting the subsequent internalization and degradation of FPN1, and was associated with ferroportin gain of function. CONCLUSIONS: The SLC40A1 p.Y333H mutation is associated with gain of function of ferroportin, representing one of the major aetiological factors of haemochromatosis in China.

Non-HFE mutations in haemochromatosis in China: combination of heterozygous mutations involving HJV signal peptide variants.

INTRODUCTION: Hereditary haemochromatosis (HH) caused by a homozygous p.C282Y mutation in haemochromatosis (HFE) gene has been well documented. However, less is known about the causative non-HFE mutation. We aimed to assess mutation patterns of haemochromatosis-related genes in Chinese patients with primary iron overload. METHODS: Patients were preanalysed for mutations in the classic HH-related genes: HFE, HJV, HAMP, TFR2 and SLC40A1. Whole exome sequencing was conducted for cases with variants in HJV signal peptide region. Representative variants were analysed for biological function. RESULTS: None of the cases analysed harboured the HFE p.C282Y; however, 21 of 22 primary iron-overload cases harboured at least one non-synonymous variant in the non-HFE genes. Specifically, p.E3D or p.Q6H variants in the HJV signal peptide region were identified in nine cases (40.9%). In two of three probands with the HJV p.E3D, exome sequencing identified accompanying variants in BMP/SMAD pathway genes, including TMPRSS6 p.T331M and BMP4 p.R269Q, and interestingly, SUGP2 p.R639Q was identified in all the three cases. Pedigree analysis showed a similar pattern of combination of heterozygous mutations in cases with HJV p.E3D or p.Q6H, with SUGP2 p.R639Q or HJV p.C321X being common mutation. In vitro siRNA interference of SUGP2 showed a novel role of downregulating the BMP/SMAD pathway. Site-directed mutagenesis of HJV p.Q6H/p.C321X in cell lines resulted in loss of membrane localisation of mutant HJV, and downregulation of p-SMAD1/5 and HAMP. CONCLUSION: Compound heterozygous mutations of HJV or combined heterozygous mutations of BMP/SMAD pathway genes, marked by HJV variants in the signal peptide region, may represent a novel aetiological factor for HH.

Regulation of Tak1 alternative splicing by splice-switching oligonucleotides.

Alternative splicing (AS) generates multiple isoforms from a single precursor mRNA, and these isoforms usually exhibit different tissue distributions and functions. Aberrant protein isoforms can lead to abnormalities in protein function and may even result in genetic disorders or cancer. In recent years, splice-switching oligonucleotides (SSOs) have emerged as a promising therapeutic strategy for several neurological diseases, but the efficacy of this strategy in other organs is less reported. In this study, we designed and synthesized SSOs targeting the splicing regulators of exon 12 of the Tak1 gene, inducing variant switching between Tak1-A and Tak1-B. We also designed SSOs capable of knockdown both Tak1 variants by inducing the aberrant splicing of exon 4. The Vivo-morpholino SSOs showed significant splice-switching of Tak1 in mouse liver, with a persistence of at least 10 days after initial SSOs delivery. Bioinformatics analysis indicated a lipid metabolism-related function for Tak1-B but not Tak1-A. The conversion of Tak1-B to Tak1-A consistently led to significant accumulation of lipids in cultured AML12 cells, as well as the dysregulation of several lipid metabolism-related genes in mouse liver. Different functional properties of the two isoforms may explain the conflicting functions previously reported for Tak1. In conclusion, our research clarified the different functions of Tak1 isoforms, and provided an efficient strategy for the functional research of the AS isoforms.

EGCG enhances the efficacy of cisplatin by downregulating hsa-miR-98-5p in NSCLC A549 cells.

In the current study, the enhanced efficacy of cisplatin caused by (-)-epigallocatechin-3-gallate (EGCG) in nonsmall cell lung cancer (NSCLC) A549 cells was observed. The tumor size was significantly smaller in vivo in the combination of cisplatin and EGCG group, as compared with cisplatin-only group. However, in NCI-H460 cells, another kind of NSCLC cells, the efficacy of cisplatin was antagonized by EGCG. MiRNA microarray showed that hsa-miR-98-5p and hsa-miR-125a-3p were differentially expressed after EGCG treatment in these 2 cell lines. After transfection of hsa-miR-98-5p inhibitor, the survival fraction of both A549 and NCI-H460 cells was decreased upon cisplatin treatment. Meanwhile, as a critical regulator in the cisplatin-induced apoptosis, p53 was elevated by silencing of hsa-miR-98-5p. These results suggested that EGCG inhibited the expression of hsa-miR-98-5p, followed by an increase of p53, thus the efficacy of cisplatin was enhanced. Bioinformatics analysis showed that hsa-miR-125a-3p might have a strong connection with classical MAPK pathway. Taken together, these findings indicate that hsa-miR-98-5p could be a potential target in clinical cisplatin treatment of NSCLC. The combination of EGCG and cisplatin might be an effective therapeutic strategy in treating some type of NSCLC, although the possibility of antagonistic interactions must also be taken into account.

Combination of low concentration of (-)-epigallocatechin gallate (EGCG) and curcumin strongly suppresses the growth of non-small cell lung cancer in vitro and in vivo through causing cell cycle arrest.

(-)-Epigallocatechin gallate (EGCG) and curcumin are two naturally derived agents that have been widely investigated worldwide. They exhibit their anti-tumor effects in many types of cancers. In the current study, the effect of the combination of the two agents on non-small cell lung cancer (NSCLC) cells was investigated. The results revealed that at low concentrations, the combination of the EGCG and curcumin strongly enhanced cell cycle arrest. Flow cytometry analysis showed that the cells were arrested at G1 and S/G2 phases. Two main cell cycle related proteins cyclin D1 and cyclin B1 were significantly inhibited at the present of EGCG and curcumin. EdU (5-ethynyl-2'-deoxyuridine) fluorescence staining showed that the DNA replication was significantly blocked. A clonal growth assay also confirmed a marked repression of cell growth. In a lung cancer xenograft node mice model, combination of EGCG and curcumin exhibited protective effect against weight loss due to tumor burden. Tumor growth was strongly repressed by the combination of the two agents, without causing any serious side-effect. Overall, these results strongly suggest that EGCG in combination with curcumin could be a candidate for chemoprevention agent of NSCLC.

[Construction of a stable centromere protein F overexpression cell model of hepatocellular carcinoma using CRISPR activation system].

Current studies have shown that centromere protein F (CENPF) was overexpressed in hepatocellular carcinoma (HCC) and might be involved in the pathogenesis of HCC. Specifically, due to the very large molecular weight (358 kDa) of CENPF full length protein, only CENPF knock-down, but not overexpression models, were applied currently to explore the carcinogenicity of CENPF in HCC. Whether CENPF overexpression is a cause or an effect in HCC remains to be illustrated. We aimed to establish a CENPF overexpression cell model using CRISPR/dCas9 synergistic activation mediator (SAM) system with lentiMPHv2 and lentiSAMv2 vectors to explore the role of CENPF overexpression in HCC. Single guide RNAs (sgRNAs) that specifically identify the transcription initiation site of CENPF gene were synthesized and inserted into the lentiSAMv2 plasmid. Huh-7 and HCCLM3 cells were first transduced with lentiMPHv2 and then selected with hygromycin B. The cells were then transduced with lentiSAMv2 carrying specific sgRNA for CENPF gene, followed by blasticidin S selection. The mRNA and protein detection results of Huh-7 and HCCLM3 cells screened by hygromycin B and blasticidin S showed that the endogenous overexpression of CENPF can be induced by sgRNA1 and sgRNA4, especially by sgRNA4. By using the CRISPR/dCas9 technique, stable cell models with overexpressed CENPF were successfully constructed to explore the role of CENPF in tumorigenesis, which provides a reference for the construction of cell models overexpressing large molecular weight protein.

DENND3 p.L708V activating variant is involved in the pathogenesis of hereditary hemochromatosis via the RAB12/TFR2 signaling pathway.

PURPOSE: Pathogenic variants in HFE and non-HFE genes have been identified in hereditary hemochromatosis (HH) in different patient populations, but there are still a considerable proportion of patients with unexplained primary iron overload. We recently identified in Chinese patients with unexplained primary iron overload a recurrent p.L708V variant in the differentially expressed in normal and neoplastic cells domain 3 (DENND3) gene, functioning as a guanine nucleotide exchange factor for small GTpase Rab12 which down-regulates TfR expression in mice. We aim to investigate the pathogenicity and the underlying mechanism of the DENND3 p.L708V variant in HH patients. METHODS: Patients with primary iron overload were analyzed for DENND3 p.L708V. TFR2 and hepcidin expression in livers were examined in HH patients harboring DENND3 p.L708V. The effects of DENND3 p.L708V on RAB12/TFR2 and downstream iron metabolic pathways were investigated in vitro and in vivo. RESULTS: Six of 31 patients with HH (19.35%) harbored the DENND3 p.L708V variant. The expression of TFR2 and hepcidin was decreased in the liver of HH patients with DENND3 p.L708V. Cells transfected with the DENND3 p.L708V vector showed up-regulation of RAB12 expression and TFR2 degradation in lysosomes, and down-regulation of the pSMAD1/5 and hepcidin. Mice models infected with adeno-associated virus expressing DENND3 p.L708V variant showed higher total serum iron concentrations and decreased HAMP level, increased amount of iron accumulation and the down-regulated of TFR2 expression in the liver. CONCLUSIONS: The DENND3 p.L708V activating variant down-regulates hepcidin expression through the DENND3/RAB12/TFR2 axis, which may represent a potential novel pathogenic factor of HH.

Identification and evaluation of novel serum autoantibody biomarkers for early diagnosis of gastric cancer and precancerous lesion.

PURPOSE: Early diagnosis is crucial for optimal prognosis of gastric cancer (GC). Hereby, we aimed to identify novel serum autoantibody-based biomarkers for precancerous lesion (PL) and early GC. METHODS: We performed serological proteome analysis (SERPA) combined with nanoliter-liquid chromatography combined with quadrupole time of flight tandem mass spectrometry (Nano-LC-Q-TOF-MS/MS) to screen for GC-associated autoantibodies. The identified autoantibodies were analyzed for potential detection value for PL and GC by enzyme-linked immunosorbent assay (ELISA). Receiver operating characteristic (ROC) curves analysis was conducted to evaluate the accuracy of the biomarkers. RESULTS: We identified seven candidates, such as mRNA export factor (RAE1), Nucleophosmin 1 (NPM1), phosphoglycerate kinase 1 (PGK1), and ADP-ribosylation factor 4 (ARF4). Antibodies against all seven proteins were present at higher levels in sera from 242 patients (51 PL, 78 early GC, 113 advanced GC) compared with sera from 122 healthy individuals. RAE1-specific autoantibody discriminated best between patients at different GC stages, with area under the curve (AUC) values of 0.710, 0.745, and 0.804 for PL, early GC, and advanced GC, respectively. Two predictive models composed of gender, RAE1, PGK1, NPM1, and ARF4 autoantibodies (Model 2 for PL) and of age, gender, RAE1, PGK1, and NPM1 autoantibodies (Model 3 for early GC) had improved diagnostic efficiencies, with AUCs of 0.803 and 0.857, sensitivities of 66.7% and 75.6%, and specificities of 78.7% and 87.7%, respectively. CONCLUSION: The identified serum tumor-associated autoantibodies (TAAbs) may have good potential for early detection of GC and PL.

Identification of novel serum autoantibody biomarkers for early esophageal squamous cell carcinoma and high-grade intraepithelial neoplasia detection.

Background: Early diagnosis of esophageal squamous cell carcinoma (ESCC) is critical for effective treatment and optimal prognosis; however, less study on serum biomarkers for the early ESCC detection has been reported. The aim of this study was to identify and evaluate several serum autoantibody biomarkers in early ESCC. Methods: We initially screened candidate tumor-associated autoantibodies (TAAbs) associated with ESCC by serological proteome analysis (SERPA) combined with nanoliter-liquid chromatography combined with quadrupole time of flight tandem mass spectrometry (nano-LC-Q-TOF-MS/MS), and the TAAbs were further subjected to analysis by Enzyme-linked immunosorbent assay (ELISA) in a clinical cohort (386 participants, including 161 patients with ESCC, 49 patients with high-grade intraepithelial neoplasia [HGIN] and 176 healthy controls [HC]). Receiver operating characteristic (ROC) curve was plotted to evaluate the diagnostic performance. Results: The serum levels of CETN2 and POFUT1 autoantibodies which were identified by SERPA were statistically different between ESCC or HGIN patients and HC in ELISA analysis with the area under the curve (AUC) values of 0.709 (95%CI: 0.654-0.764) and 0.741 (95%CI: 0.689-0.793), 0.717 (95%CI: 0.634-0.800) and 0.703 (95%CI: 0.627-0.779) for detection of ESCC and HGIN, respectively. Combining these two markers, the AUCs were 0.781 (95%CI: 0.733-0.829), 0.754 (95%CI: 0.694-0.814) and 0.756 (95%CI: 0.686-0.827) when distinguishing ESCC, early ESCC and HGIN from HC, respectively. Meanwhile, the expression of CETN2 and POFUT1 was found to be correlated with ESCC progression. Conclusions: Our data suggest that CETN2 and POFUT1 autoantibodies have potential diagnostic value for ESCC and HGIN, which may provide novel insights for early ESCC and precancerous lesions detection.

[Construction and analysis of transcriptome-based hepatolenticular degeneration regulatory network].

A transcriptional regulatory network for wild-type and ATP7B-knockout HepG2 cells exposed to copper was constructed by bioinformatics methods to explore the potential mechanism of key transcription factors in the pathogenesis of hepatolenticular degeneration. The differentially expressed genes (DEGs) for wild-type and ATP7B-knockout HepG2 cell lines without copper and exposed to copper were collected from the gene expression omnibus (GEO) database. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis were performed for DEGs induced by copper. The key functional modules and genes were identified based on the protein-protein interaction (PPI) network. Moreover, the enrichment analysis of genes in functional modules was performed. Finally, a transcriptional regulatory network was constructed to screen the core transcription factors. A total of 1 034 genes, including 509 down-regulated genes and 525 up-regulated genes, were selected as DEGs. The up-regulated and down-regulated functional modules based on PPI network included 3 785 and 3 931 genes, respectively. Genes in key functional modules were enriched in cell-substrate junction, chromosomal region, spliceosomal complex and ribosome. They were involved in mRNA processing, histone modification, RNA splicing, regulation of DNA metabolic process, protein phosphorylation and other biological processes. Moreover, they were correlated to transcriptional coregulator activity, DNA-binding transcription factor binding, ubiquitin-like protein ligase binding and other molecular functions. KEGG analysis showed that genes in key functional modules were significantly enriched in hepatitis B, MAPK signaling pathway, cellular senescence and apoptosis, neurotrophin signaling pathway and pathways of neurodegeneration-multiple diseases. The transcriptional regulatory network contained 11 differentially expressed transcription factors and 96 DEGs. Among them, U2AF1, NFRKB, FUS, MAX, SRSF1, CEBPA and RXRA were the core transcription factors, which may facilitate the study of the biological function of relevant molecules in transcriptional regulation of hepatolenticular degeneration.

Identification of novel non-HFE mutations in Chinese patients with hereditary hemochromatosis.

BACKGROUNDS: Hereditary hemochromatosis (HH) is mainly caused by homozygous p.C282Y mutations in HFE in the Caucasians. We recently reported non-HFE mutations constitute the major cause of HH in Chinese. However, there is still a relatively high proportion of cases with primary iron overload from unexplained causes. We aimed to explore novel non-HFE mutations in Chinese patients with primary iron overload. METHODS: Whole exome sequence was conducted to screen mutations in novel HH-related genes in the 9 cases with unexplained primary iron overload. Then the representative candidate genes were screened for mutations in another cohort of 18 HH cases. The biological function of the selected genes and variants were analyzed in vitro. RESULTS: Whole exome sequencing of 9 cases with unexplained primary iron overload identified 42 missense variants in 40 genes associated with iron metabolism pathway genes such as UBE2O p.K689R and PCSK7 p.R711W. Subsequent Sanger sequencing of the UBE2O and PCSK7 genes in the 27 cases with primary iron overload identified p.K689R in UBE2O, p.R711W and p.V143F in PCSK7 at frequency of 2/27,1/27 and 2/27 respectively. In vitro siRNA interference of UBE2O and PCSK7 resulted in down-regulated HAMP mRNA expression. Adenovirus generation of UBE2O p.K689R in cell lines resulted in increased expression of SMAD6 and SMAD7 and downregulation of p-SMAD1/5 and HAMP expression, and the reduction of hepcidin level. CONCLUSIONS: Our study identified a series of novel candidate non-HFE mutations in Chinese patients with HH. These may provide insights into the genetic basis of unexplained primary iron overload.

Identification of potential modifier genes in Chinese patients with Wilson disease.

The mutations in modifier genes may contribute to some inherited diseases including Wilson disease (WD). This study was designed to identify potential modifier genes that contribute to WD. A total of 10 WD patients with single or no heterozygous ATP7B mutations were recruited for whole-exome sequencing (WES). Five hundred and thirteen candidate genes, of which the genetic variants present in at least two patients, were identified. In order to clarify which proteins might be involved in copper transfer or metabolism processes, the isobaric tags for relative and absolute quantitation (iTRAQ) was performed to identify the differentially expressed proteins between normal and CuSO4-treated cell lines. Thirteen genes/proteins were identified by both WES and iTRAQ, indicating that disease-causing variants of these genes may actually contribute to the aberrant copper ion accumulation. Additionally, the c.86C > T (p.S29L) mutation in the SLC31A2 gene (coding CTR2) has a relative higher frequency in our cohort of WD patients (6/191) than reported (0.0024 in gnomAD database) in our healthy donors (0/109), and CTR2S29L leads to increased intracellular Cu concentration and Cu-induced apoptosis in cultured cell lines. In conclusion, the WES and iTRAQ approaches successfully identified several disease-causing variants in potential modifier genes that may be involved in the WD phenotype.

Aldehyde dehydrogenase 1B1 is a potential marker of colorectal tumors.

Colorectal cancer (CRC) is common worldwide. Aldehyde dehydrogenase 1B1 (ALDH1B1), a member of the ALDH1 family, serves as a biomarker for cancer stem cells. We hypothesized that ALDH1B1 expression is associated with colorectal tumors. Immunohistochemistry was used to detect ALDH1B1 expression across a commercial colorectal tissue microarray. The signal intensities of the positively stained tissues were expressed using the mean integrated optical density (mean IOD). We also analyzed ALDH1B1 mRNA expression in the Oncomine database. The associations between ALDH1B1 expression and CRC stage and prognosis were then evaluated using the web-based tools, GEPIA and UALCAN. Analysis of the tissue microarray revealed that the expression of ALDH1B1 was significantly higher in colorectal adenomas and colorectal adenocarcinoma (IOD/area values=0.117±0.070 and 0.168±0.0168, respectively) compared with normal and cancer-adjacent tissues (IOD/area values=0.051±0.028 and 0.068±0.053). For samples collected in the hospital, ALDH1B1 was highly expressed in the adenoma (IOD/area=0.103±0.054) and CRC (IOD/area=0.116±0.059) tissues compared with the cancer-adjacent tissues (IOD/area=0.066±0.024, p<0.05). The expression of ALDH1B1 in tissues from two resources was not found to be significantly associated with CRC stage. In Oncomine, ALDH1B1 mRNA expression was increased in the colorectal tumor tissues compared with the normal colorectal tissues (p=0.024) and its expression was independent of CRC stage and prognosis (p<0.05). Thus, while the protein and mRNA expression of ALDH1B1 suggests that it is a potential marker of colorectal tumors, its expression is independent of CRC stage and prognosis.

Apoptosis-induced translocation of centromere protein F in its corresponding autoantibody production in hepatocellular carcinoma.

Serum autoantibodies against tumor-associated antigen have important value in the early diagnosis of hepatocellular carcinoma (HCC), but the mechanism of autoantibody production is poorly understood. We previously showed that autoantibodies against the centromere protein F (CENPF) may be useful as an early diagnostic marker for HCC. Here we explored the mechanism of cell apoptosis-based CENPF autoantibody production and verified the correlation of CENPF autoantibody level with HCC development. We demonstrated that CENPF was overexpressed and aberrantly localized throughout the nuclei and cytoplasm in human HCC cells compared with hepatic cells. CENPF overexpression promoted the production of CENPF autoantibodies in a manner that correlated with tumor growth of mouse HCC model. During apoptosis of HCC cells, CENPF protein translocated to apoptotic vesicles and relocalized at the cell surface. Through isolating apoptotic components, we found apoptotic body and blebs with lower CD31 and CD47 expression more effectively induced DC phagocytosis and maturation compared with apoptotic intact cells in vitro, and this DC response was independent of CENPF expression. Moreover, injection of mice with apoptotic bodies and blebs effectively induced an immune response and the production of CENPF-specific antibodies. Our findings provide a first elucidation of mechanisms underlying the CENPF autoantibody production via cell apoptosis-induced CENPF translocation, and demonstrate a direct correlation between CENPF autoantibody levels and HCC progression, suggesting the potential of CENPF autoantibody as an HCC diagnostic marker.

Role of tight junction-associated MARVEL protein marvelD3 in migration and epithelial-mesenchymal transition of hepatocellular carcinoma.

MarvelD3, a recently identified tight junction membrane protein, could be associated with hepatocellular carcinoma (HCC). We aimed to investigate the role of marvelD3 in Epithelial-Mesenchymal Transition (EMT) and migration of HCC and explore the underlying molecular mechanisms. First, we assessed marvlD3 expression in HCC and normal liver tissues and found loss of marvelD3 expression was significantly correlated with the occurrence and TNM stage of HCC. Second, we detected that marvelD3 was downregulated in HCC cells with transforming growth factor ß1 and snail/slug-induced EMT. Finally, we analyzed expression of marvelD3 protein was significantly associated with EMT and the NF-κB signaling pathway. Our study demonstrated that MarvelD3 inhibited EMT and migration of HCC cells along with inhibiting NF-κB signaling pathway.Abbreviations:HCC, Hepatocellular carcinoma; TJ, Tight junction; MARVEL, MAL and related proteins for vesicle trafficking and membrane link; EMT, Epithelial-mesenchymal transition; NF-κB, Nuclear factor kappa B; TAMPs, Tight junction-associated marvel proteins; TGF-ß1, Transforming growth factor-ß1; MMP9, matrix metallopeptidase 9; RT-PCR, Real-time PCR; IHC, Immunohistochemistry; IF, Immunofluorescence.

Correlation of genotype and phenotype in 32 patients with hereditary hemochromatosis in China.

BACKGROUND: Hereditary hemochromatosis (HH) is widely recognized and clinical manifestations of hemochromatosis-related (HFE-related) HH is well studied in European populations. Less is known about the clinical and laboratory characteristics of non-HFE related HH in Asian population. We aimed to explore the relationship between genotype and clinical phenotype in Chinese patients with non-HFE related hereditary hemochromatosis. METHODS: Peripheral blood samples and clinical data of patients with primary iron overload were collected from the China Registry of Genetic/Metabolic Liver Diseases. Sanger sequencing was performed in cases with primary iron overload, for 5 known HH related genes (HFE, HJV, HAMP, TFR2 and SLC40A1) and 2 novel iron homeostasis-related genes (DENND3 and SUGP2). The correlation of genotype and clinical phenotype in these patients was analyzed. RESULTS: Of the 32 patients with primary iron overload (23 were males and 9 were females), non-HFE variants were detected in 31 (31/32, 97%), including 8 pathogenic variants in HJV, 7 pathogenic variants in SLC40A1, 8 likely pathogenic variants in SUGP2 and 5 likely pathogenic variants in DENND3 cases. Among these 31 cases, 4 cases harbored homozygous variants, 2 cases harbored homozygous + heterozygous variants, 19 cases harbored heterozygous or combined heterozygous variants, and 6 cases harbored no any damaging variants. None of investigated cases carried damaging HAMP and TFR2 variants were found. 8 cases were classified as type 2A HH and 6 cases as type 4 HH, 10 cases as non-classical genotype, and 6 cases had no pathogenic variants from 31 cases. During the statistical analysis, we excluded one case (SLC40A1 IVS3 + 10delGTT + SUGP2 p. R639Q(homo)) with difficulty in grouping due to combined damaging variants. Cases with type 2A HH have an earlier age at diagnosis (p = 0.007). The iron index of cases in type 2A HH and type 4 HH was higher than that in other groups (p = 0.01). Arthropathy was relatively rare in all groups. None of cases with type 2A HH developed cirrhosis. Cirrhosis and diabetes are more prevalent in type 4 HH. The incidence of cirrhosis (p = 0.011), cardiac involvement (p = 0.042), diabetes (p = 0.035) and hypogonadism (p = 0.020) was statistically significant in the four groups. However, due to the limited sample size, the pairwise comparison showed no significant difference. CONCLUSIONS: This is the first comprehensive analysis about the gene variant spectrum and phenotypic aspects of non-HFE HH in China. The results will be useful to the identification, diagnosis and management of HH in China.