Comprehensive and integrative analysis of hepatocellular carcinoma (HCC) is important. In this study, we explored Taiwanese HCCs using multi-omics analyses.We analyzed 254 HCCs by whole genome sequencing and total RNA sequencing, and then used bioinformatic tools to analyze genomic and transcriptomic alterations in coding and non-coding sequences to explore the clinical importance of each sequence.TERT, TP53, CTNNB1, RB1, and ARID1A. Genetic alteration frequencies influenced the etiology of HCC; some alterations were also correlated with clinicopathological conditions. Many cancer-related genes had copy number alterations (CNAs) and structure variants (SVs) that changed according to etiology and exhibited potential associations with survival. We also identified several alterations in histone-related genes, HCC-related long non-coding RNAs, and non-coding driver genes that may contribute to the onset and progression of HCC. Transcriptomic analysis revealed that 229 differentially expressed and 148 novel alternative splicing (AS) genes, as well as the presence of fusion genes, were associated with patient survival. Moreover, somatic mutations, CNAs, and SVs were associated with immune checkpoint gene expression and tumor microenvironment. Finally, we identified relationships among AS, immune checkpoint gene expression and tumor microenvironment.The frequencies of the five most commonly mutated cancer-related genes were This study shows that genomic alterations are associated with survival, including DNA-based and RNA-based data. Moreover, genomic alterations and their associations with immune checkpoint genes and the tumor microenvironment may provide novel insights for the diagnosis and treatment of HCC.The online version contains supplementary material available at 10.1186/s40364-023-00492-7. TP53, CTNNB1, and TERT) [Liver cancer, of which hepatocellular carcinoma (HCC) is the predominant form, is the fifth most common cancer and the second most common cause of cancer-related death worldwide . In addind TERT) , 8–18. Dnd TERT) , 20.Since 1984, the Taiwanese government has introduced a series of national initiatives to prevent hepatitis B virus (HBV)-related HCC through universal HBV vaccination. These efforts have resulted in a reduced incidence of HBV-related HCC in the young population . ChineseHCC was identified by pathological diagnosis. Tumor and adjacent non-tumor liver tissue samples were collected after surgical resection, then frozen at − 80 °C and stored at the Tissue Bank of China Medical University Hospital (CMUH). all specimens were obtained from patients with appropriate consent from the relevant Institutional Review Board . DNA and RNA were collected from samples using the Mini (Qiagen) and NucleoSpin (Macherey-Nagel) kit, respectively. We used commercial technology for WGS and RNA-seq. Data were analyzed using the Illumina DRAGEN Bio-IT Platform. Detailed methods are presented in the supplementary material.The demographic data of the 254 HCC patients are presented in Table TP53 (22% vs. 28%), CTNNB1 (13% vs. 24%), MUC16 (8% vs. 16%), LRP1B (6% vs. 8%), ALB (5% vs. 11%), and CSMD3 (5% vs. 8%) and the overall mutation rate was lower than the rate reported in The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC). Some mutated genes were present at higher frequencies, such as RB1 (11% vs. 5%), ARID1A (10% vs. 7%), AXIN1 (9% vs. 8%), and ARID2 (8% vs. 5%), compared with TCGA-LIHC; the TERT mutation rate (47.24%) was determined by analysis of promoter , ATRX was more frequently mutated in HBV-related HCC (p = 0.0367), COL3A1 was more frequently mutated in dual HBV/HCV inflection (p = 0.0061), and CSMD3 was more frequently mutated in double cancer-related HCC (p = 0.0104)  and were uncommon in HBV-related HCC (p = 2.812e-06). Moreover, the presence of TERT promoter mutation was associated with a significant decrease in overall survival (p = 0.039) , and HCV-related HCC was associated with a higher rate of cirrhosis (p = 2.961e-05). Kaplan-Meier survival analysis indicated that the different etiologies did not influence survival. Fig. . TERT pr39) Fig. .CHD3, GPS2, PER1, and YWHAE in the17p deletion) [CNAs, which involved chromosomal regions and related genes, are shown in the heatmap Fig.  and circeletion) , 25 was (SVs) revealed 17,639 somatic SVs in our cohort  had the highest mutated rate  had more mutations  or CADD ≥ 30 (21 cases); te Table . We valiing Fig. . Among 7ns Table . Among 3ts Table .TERT mutation are shown in Fig. We performed mutational signature analysis on the core set of 254 HCCs by non-negative matrix factorization. There were three types of mutational signatures in Taiwanese HCCs. The case numbers and distributions of mutational signatures in each cluster are shown in Fig. Greater proportions of HCV and dual HBV/HCV infection-related HCCs exhibited the AA exposure signature ; this signature was present in 9/106, 0/28, and 0/19 of HBV, non-HBV/non-HCV, and double cancer-related HCC, respectively. Among cases of non-HBV/non-C and double cancer-related HCCs, 6/28 and 8/19, respectively, exhibited the chemotherapy-related signature SBS25; no cases with other etiologies exhibited SBS25 Fig. B.details can be provided on request). Next, we analyzed the DEGs and their associations with survival; 229 genes (123 protein-coding and 106 non-coding) were associated with survival. Among the 123 protein-coding genes, increased expression levels of 28 genes were associated with better survival, whereas increased expression levels of the remaining 95 genes were correlated with worse survival. Among the 106 non-coding genes, increased expression levels of 33 genes (including 16 novel genes) and the remaining 73 genes (including 29 novel genes) were associated with better or worse survival, respectively   Table .IGF2BP3, PKIA, ANKS1B, AL049734.2, and AC072028.1) that were significantly overexpressed in females (p < 0.001)  Table .SLC45A2-AMACR, ITCH-ASIP, and RNF138-RNF125) were present in > 1 case . Among the 111 novel fusion genes, 18 involving known cancer genes  (Table Fifty of fusion genes have been reported and 14 involving known cancer genes (1) Table .We used kraken tools to analyze HBV DNAs and HBV-related transcripts. In total, 116 HBV-related HCCs were closely associated with the results of serum testing, RNA-seq, and WGS; 131 cases were closely associated with the results of WGS and RNA-seq; and 3 and 12 cases were closely associated with only WGS or only RNA-seq, respectively Fig. A.KMT2B, TERT, and CC2. Only a few DNA fusion genes had RNA transcripts  values for Taiwanese HCCs. There were 914 AS events for 0.2 PSI and 920 events for 0.3 PSI. For both 0.2 and 0.3 PSI, the number of common events was 345 Fig. . We thenCDK13, CFLAR, EGLN1, and ZNF717 exhibited aberrant effects on survival that were caused by different types of AS event  were selected for analysis (Details can be provided on request). Only mutations in ARID1A was associated with higher expression of CD70 . Statistically significant copy changes occurred in AKT1 (influencing TNFSF14), AKT2 (influencing CD40LG), ARID1A (influencing CD276), ARID1B (influencing CD40LG and TNFRSF4), AXIN1 , GNAS (influencing TNFSF14 and PDCD1LG2), IDH2 (influencing CD274 and CD96), NF1 (influencing CD40LG), SMARC4 , STAT3  and TSC2 (influencing CD276 and TRAC)  Fig. .CTNNB1 mutations were associated with a decrease in epithelial cells but increases in hematopoietic stem cells and megakaryocyte-erythroid progenitor (MEP). RB1 mutations and copy loss were associated with decreases in adipocytes, lymphatic (ly) endothelial cells, stroma score, and preadipocytes; they were associated with increases in common lymphoid progenitor (CLP), pro B-cells, and Th1 and Th2 cells. TERT mutations were associated with a decrease in neurons, whereas ARID1B copy loss was associated with an increase in microvascular (mv) endothelial cells. IL6ST copy loss was associated with a decrease in microenvironment score. MDM4 amplification was associated with a decrease in pro B-cells, whereas TSC1 amplification was associated with a decrease in adipocytes. TSC1 copy loss was associated with an increase in sebocytes. AXIN1, BRD7, IDH2 and TSC2 copy loss were each associated with a decrease in hepatocytes  to explore their associations with the numbers of immune and stroma cell. We found that tes Fig. .With respect to 17p deletion and 7q duplication, none remained statistically significant after adjustment. The SVs (AnnotSV_ID: 1_105963115_182721369_DUP_1) involving 38 protein genes was associated with basophils Fig. .Associations among the expression of immune checkpoint genes, the number of 64 cell types/3 scores, and AS.TNFRSF4, TNFRSF18, VSIR, TMIGD2, ICOSLG, TNFRSF14, PDCD1, and CD70, along with decreases in the expression levels of TNFSF4, CEACAM1, PDCD1LG2, and CD86; AS of other genes showed similar associations  was associated with larger changes in the numbers of mesenchymal stem cells (MSC), natural killer T (NKT), eosinophils, central memory CD4 + T cell (CD4 + Tcm), pericytes, ly endothelial cells, mv endothelial cells, Th1 cells, MEP, endothelial cells, and inflammatory (M1) macrophages, as well as greater changes in stroma score; it was associated with smaller changes in the numbers of CD8 + naïve T-cells, CLP, Th2 cells, and smooth muscle. This AS was also associated with significant increases in the expression levels of the immune checkpoint genes LINC473 and HAGLR had a higher mutation rate, as indicated by highly stringent selection. Three cases had non-coding driver gene mutations. We also found that 120 cases had TERT promoter mutations. These non-coding genes may play a role in the onset and progression of Taiwanese HCCs.In this study, we used WGS to analyze a large number of HCC cases and confirm the uniqueness of Taiwanese HCCs implied in our previous study , 32. We HILS1 had a higher mutated rate, as indicated by highly stringent selection. Thus, we suspect that histone mutations contribute to the onset and progression of HCC [Recently, the roles of histone gene mutations in cancer onset and progression have been clarified ; howevern of HCC , 34.AURKB, CHD3 (TSG), GAS7, GPS2 (TSG), PER1 (TSG), TP53, RABEP1, USP6 (oncogene), and YWHAE (TSG) resulting in both oncogene and TSG deletions that are associated with better survival. Our results indicate that the analysis of alterations (including bases and copy numbers) in each oncogene or TSG does not accurately assess the clinical impacts; direct evaluation of large CNAs is needed to improve the overall understanding of HCC onset and progression.Each case of HCC usually had many alterations of large CNAs; these CNAs may contain several genes , resulting in simultaneous gain or loss of these genes. For example, deletion of 17p includes In SV analysis, we used pLI methods, Taiwan biobank and gnomAD databases, and non-tumor tissues and benign liver lesions to select somatic SVs. Using this approach, we identified many HCC-related somatic SVs; 13 unique SVs were correlated with patient survival.CCNA2 fusions, defining a homogenous entity of aggressive HCC [CCNA2 fusion in this study, we identified that HBV inserted into CCNA2 and CCNE1.We also analyzed fusion genes and found that they involve known cancer genes whether they are already reported fusions or novel fusions. Bayard et al., indicated that some HCC subgroups exhibit cyclin activation through various mechanisms, including HBV and adeno-associated virus type 2 insertions and enhancer hijacking and recurrent sive HCC . AlthougARID1A led to high levels of CD70 expression.Expression patterns of immune checkpoint genes in tumor cells have important role in evading attacks by host immune cells; mutations in cancer-related genes may influence these processes . Our resRB1 mutations and copy loss were associated with decreases in adipocytes, ly endothelial cells, stromal score, and preadipocytes; they were associated with increases in CLP, pro B-cells, and Th1 and Th2 cells. The SV (AnnotSV_ID: 1_105963115_182721369_DUP_1) was associated with a decrease in basophils. Accordingly, we suspect that genomic alterations have important roles in the HCC microenvironment [Diversity in the immune cell population and tumor microenvironment and associations with clinical outcomes have been identified in many studies –41. Mostironment .KCMF1 oncogene was associated with increases in the numbers of MSC, NKT, eosinophils, CD4 + Tcm, pericytes, and ly and mv endothelial cells; it was associated with decreases in the numbers of CD8 + naïve T-cells, CLP, Th2 cells, and smooth muscle; and it was associated with upregulation of TNSRSF4, TNSRSF18, and VSIR, as well as downregulation of TNFSF4 and CEACAM1. Some AS patterns have already been associated with immune cell behavior; example associations include NCKIPSD (SPIN90) with stromal fibroblast activation in low stiffness stroma [HSD3B7 with T cell-dependent plasma cell responses [DAPK1 with NK cell ability and CD8 + T cell activation [HSPA12B with tumor-associated endothelial cell activity and M2 polarization [In terms of the effects of survival-related AS events on immune cells and the expression patterns of immune checkpoint genes, AS of cancer-related gene had greater effects on immune and stroma cells, relative to AS without these characteristics; the numbers of immune and stroma cells were associated with both survival-related AS and the expression patterns of immune checkpoint genes. For example, AS that resulted in the loss of the s stroma , HSD3B7 esponses , DAPK1 wtivation , and HSPrization . Based orization , 47.Our study makes several significant contributions to the field of HCC research. Firstly, by conducting a comprehensive analysis of data that includes WGS and RNA-seq from a large number of HCCs, our study provides a more complete understanding of the genetic and transcriptomic landscape of HCC. Secondly, we explored the relationships among immune checkpoint gene expression, the tumor microenvironment, and AS, and our findings suggest that AS could be a promising approach for HCC treatment. Thirdly, we have reconfirmed the uniqueness of HCCs in Taiwanese patients.A notable limitation of this study was that it used stringent criteria to select HCC-related genomic alterations, which may have resulted in the exclusion of some rare or weak effective alterations. Additionally, all cases were surgically treated, and we excluded patients who underwent transplantations as this may have resulted in different survival times compared to other studies. Finally, we used total RNA for transcriptome analysis, which may result in the loss of genes with low expression levels.In summary, we performed multi-omics analysis in HCC patients. CNAs, SVs, expression levels, alternative transcripts, and fusion transcripts could serve as potential biomarker for HCC. Overall, comprehensive and integrated approaches will provide a better understanding of the molecular basis of HCC and may facilitate the development of new therapeutic strategies.Below is the link to the electronic supplementary material.Additional file 1: Figure S1. Survival curve indicating the effect of the HCC treatment approach on patient survival.


TROP2, that stands out for its expression in the majority of solid tumors in human, for its impact on the prognosis of most solid cancers and for its role as driver of cancer growth and metastatic diffusion, through overexpression as a wild-type form. The Trop-2 signaling network encompasses CREB1, Jun, NF-κB, Rb, STAT1 and STAT3, through induction of cyclin D1 and MAPK/ERK. Notably, Trop-2-driven pathways vastly overlap with those activated by most functionally relevant/most frequently mutated RAS and TP53, and are co-expressed in a large fraction of individual tumor cases, suggesting functional overlap. Mutated Ras was shown to synergize with the TROP2-CYCLIND1 mRNA chimera in transforming primary cells into tumorigenic ones. Genomic loss of TROP2 was found to promote carcinogenesis in squamous cell carcinomas through modulation of Src and mutated Ras pathways. DNA methylation and TP53 status were shown to cause genome instability and TROP gene amplification, together with Trop-2 protein overexpression. These findings suggest that mutagenic and the TROP2 non-mutagenic pathways deeply intertwine in driving transformed cell growth and malignant progression of solid cancers.The advent of high throughput DNA sequencing is providing massive amounts of tumor-associated mutation data. Implicit in these analyses is the assumption that, by acquiring a series of hallmark changes, normal cells evolve along a neoplastic path. However, the lack of correlation between cancer risk and global exposure to mutagenic factors provides arguments against this model. This suggested that additional, non-mutagenic factors are at work in cancer development. A candidate determinant is  TheCAM gene , and a tCAM gene , and twoCAM gene .TROP2 mRNA expression levels in normal tissues are shown in in vivo  gene was shown to promote carcinogenesis in squamous cell carcinomas through modulation of Arf, Src and mutated Ras pathways in mTrop2 mouse knockouts   , while oy, GDLD)  and worly, GDLD) . Howevery, GDLD) , and novy, GDLD) . No cleay, GDLD) .2.3TROP2 gene was found to be under epigenetic control in choriocarcinomas, i.e. trophoblast-derived malignancies ,-epigallocatechin-3-gallate (EGCG) affected the post-transcriptional processing of the  protein .2.5TROP gene copy number variation, with corresponding additional alteration of protein expression levels . Ras-transformed keratinocytes derived from mTrop2(-/-)Arf(-/-) mice exhibit enhanced proliferative and migratory capacity as well as increased activation of MAPK and Src (mTrop2(-/-) keratinocytes were shown to undergo epithelial to mesenchymal transition (EMT) and formed tumors with spindle cell histology. Consistent with this, TROP2 mRNA levels were found to be down-regulated in head and neck squamous cell carcinomas undergoing EMT   , though s for PC Figure 3pathways  at earlypathways . Along Phe cases , suggestCACNA1G, CDH1, CDKN2A, DAPK1, MGMT, MINT1-2-31-32, MLH1, RARB, THBS1 and TIMP3  can be found in the article/supplementary material.SA designed the strategy of the article. EG, RDP, GS, SA contributed to data collection, writing of the article and revision of the final text. All authors contributed to the article and approved the submitted version.


CRELD2, a member of the cysteine-rich epidermal growth factor-like domain (CRELD) protein family, is both an endoplasmic reticulum (ER)-resident protein and a secretory factor. The expression and secretion of CRELD2 are dramatically induced by ER stress. CRELD2 is ubiquitously expressed in multiple tissues at different levels, suggesting its crucial and diverse roles in different tissues. Recent studies suggest that CRELD2 is associated with cartilage/bone metabolism homeostasis and pathological conditions involving ER stress such as chronic liver diseases, cardiovascular diseases, kidney diseases, and cancer. Herein, we first summarize ER stress and then critically review recent advances in the knowledge of the characteristics and functions of CRELD2 in various human diseases. Furthermore, we highlight challenges and present future directions to elucidate the roles of CRELD2 in human health and disease. The ECRELD1, the funding member of this family, is known as the AVSD2 gene as mutations in CRELD1 are associated with cardiac atrioventricular septal defects (AVSD)  –26. CRELs (AVSD) . Both CRs (AVSD) , 17, 28.s (AVSD) . Functios (AVSD) –32. In cs (AVSD) . To date2The ER is an important organelle in eukaryotic cells that plays essential roles in orchestrating the homeostasis of at least one-third of the proteins within a cell and ensuring cell survival , 33. ER In mammals, the UPR comprises three signaling pathways initiated by three ER transmembrane sensors: inositol-requiring enzyme 1 (IRE1α), protein kinase R (PKR)-like ER kinase (PERK), and activating transcription factor 6 (ATF6) –36. UndeXBP1 mRNA through its endonuclease activity, generating a frameshift when translating the spliced XBP1 (XBP1s) isoform transcript  transcription factor XBP1s, which regulates the transcription of multiple genes that are implicated in diverse biological pathways such as those that function in ER-associated protein degradation (ERAD) and folding proteins  and kinase activities –41. Uponanscript –44. XBP1proteins –39, 43. proteins –49. Moreproteins , 51  and site-2 protease (S2P), releasing an active ATF6 transcription factor fragment (ATFp50)  –56. This(ATFp50) –59   . Sequencδ, ϵ, ζ) . In contδ, ϵ, ζ) , 28. CREn CRELD2 . NotablyD2α gene .CRELD family, CRELD1 and CRELD2 exhibit 51% homology in the nucleic acid sequences of the coding regions. At the amino acid level, they were 38% identical and 51% similar , which is highly conserved among different species , tunicamycin , and brefeldin A   . Further stimuli .Creld2, as well as several other genes involved in ER stress. This upregulation was significantly attenuated by salubrinal and its derivatives, which are the inhibitors of ER stress , drastically suppressed the spontaneous secretion of CRELD2. Sar1 plays an essential role in early step in vesicle budding and regulating vesicular transport from the ER to the Golgi  and collagen type X alpha 1 chain (Col10a1) mutant growth plates, CRELD2 was also detectable at significant levels in the extracellular matrix (ECM) but not in wild-type controls EDL in the C-terminal region of CRELD2 regulates its secretion. For example, in HEK293 cells, the modification of the C-terminal region, such as the deletion of four C-terminal amino acids from mouse CRELD2 or addition of tag-peptides to its C-terminus, drastically enhanced CRELD2 secretion . Additioaffinity , 77–84. affinity . Under naffinity , 85. Thuaffinity , 16  and alcoholic liver diseases , 111–116he liver . Subsequhe liver . Moreovedetected . HoweverCreld2 knockout (Creld2-/-) mouse model to investigate CRELD2 function in vivo (Creld2-/- mice exhibited a reduction in body weight under both a chow diet and high-fat diet (HFD) compared to their controls but developed insulin resistance. Additionally, Creld2-/- livers generally contained fewer lipids, which could account for lower body weight. Notably, their results clearly showed that 12 weeks of HFD did not induce ER stress in the liver, and neither transcriptome nor protein expression analysis revealed a lipid-driven ER stress response in the livers of Creld2-/- mice. Moreover, this study revealed that the adipose weights of Creld2-/- mice were significantly reduced ; however, expression in female patients remained unaffected . Notablyteatosis . Based o4.2Creld2, accompanied by other UPR genes, and this upregulation was remarkably reversed by treatment with salubrinal, which is an ER stress inhibitor , which is a clinically variable and genetically heterogeneous chondrodysplasia characterized by mild short stature, joint pain, and stiffness and early onset osteoarthritis   . Notably the ECM . Moreove the ECM . Additio -stimulated osteogenic differentiation of mesenchymal stem cells , a master regulator of osteoclastogenesis  in the promoter sequences of Creld2, and CRELD2 depletion suppresses tumor progression, indicating that the paracrine ROCK-PERK-ATF4-CRELD2 axis promotes breast cancer progression. They also revealed that high CRELD2 expression was associated with progressive human breast cancer , due to mutations in the omodulin , 125–127omodulin . Additioomodulin . These domodulin . This im5in vitro and in vivo studies. Expression pattern analysis revealed that CRELD2 is ubiquitously expressed in multiple tissues to different extents, suggesting that CRELD2 may play important but diverse roles in different organs, tissues, and in different cell types. Moreover, CRELD2 is associated with various diseases and has crucial roles in various physiological and pathological processes, including liver metabolism homeostasis, cartilage and bone metabolism, and cancer progression and survival, as well as potential role as a biomarker for kidney diseases. However, despite significant progress, many outstanding concerns regarding CRELD2 remain unaddressed.Over the past decades, increasing evidence has revealed the characteristics and functions of CRELD2. Molecular studies have demonstrated that CRELD2 acts as both an ER-resident protein and a secretory factor. Various cellular stimuli, particularly ER stress, remarkably enhance CRELD2 expression and secretion, as demonstrated by multiple First, multiple isoforms of human CRELD2 protein cause variations in their subcellular localization, expression patterns, and functions, which have not yet been comprehensively elucidated. In addition, although there is evidence regarding the mechanism through which ER stress induces CRELD2 expression, it remains unclear whether other mechanisms are also involved in this process. Additional to the upregulation of CRELD2 expression, ER stress can drastically enhance its secretion. However, information on the changes in circulating CRELD2 levels in ER stress-related diseases, including diabetes, obesity, inflammation, NAFLD, CVDs, kidney diseases, and cancer, is still lacking. The source of circulating CRELD2 in these pathological conditions also remains unknown. Moreover, MANF overexpression significantly increased CRELD2 secretion. However, the exact mechanism underlying this process remains unclear. Whether CRELD2 plays a role in the function of MANF and whether there exists any link between these two proteins also remain unknown and require further studies. Similar to MANF, CRELD2 may possess a dual-function model (intracellular and extracellular functions). However, knowledge about its functions and mechanisms, particularly that of extracellular CRELD2, is still lacking. Current evidence has revealed an association between CRELD2 and various human diseases, whereas the direct causal link between CRELD2 and the pathogenesis of diseases remains poorly understood. Further investigations may provide clues as to whether the addition of exogenous CRELD2 or blocking endogenous CRELD2 is therapeutically beneficial for human diseases. This should also be validated in mouse models using CRELD2 peptides or specific antibodies. Addressing these gaps in knowledge would be beneficial for uncovering the pathophysiological significance of CRELD2 and its potential clinical applications.JH contributed to the guidance of this review and gave the final approval of the version to be published. QT drafted the manuscript. QL, YL, and LM prepared the figures. All authors read and approved the final manuscript.


Dental mesenchymal stem cells (DMSCs) are multipotent progenitor cells that can differentiate into multiple lineages including odontoblasts, osteoblasts, chondrocytes, neural cells, myocytes, cardiomyocytes, adipocytes, endothelial cells, melanocytes, and hepatocytes. Odontoblastic differentiation of DMSCs is pivotal in dentinogenesis, a delicate and dynamic process regulated at the molecular level by signaling pathways, transcription factors, and posttranscriptional and epigenetic regulation. Mutations or dysregulation of related genes may contribute to genetic diseases with dentin defects caused by impaired odontoblastic differentiation, including tricho-dento-osseous (TDO) syndrome, X-linked hypophosphatemic rickets (XLH), Raine syndrome (RS), hypophosphatasia (HPP), Schimke immuno-osseous dysplasia (SIOD), and Elsahy-Waters syndrome (EWS). Herein, recent progress in the molecular regulation of the odontoblastic differentiation of DMSCs is summarized. In addition, genetic syndromes associated with disorders of odontoblastic differentiation of DMSCs are discussed. An improved understanding of the molecular regulation and related genetic syndromes may help clinicians better understand the etiology and pathogenesis of dentin lesions in systematic diseases and identify novel treatment targets. Dentin is a thick and highly mineralized tissue layer under the enamel that protects the dental pulp cavity from infections, supports and provides nutrition to the enamel, and alleviates dental pressure . They arin vivo of SHEDs is higher. However, a complete dentin-pulp-like complex cannot be reconstituted using SHEDs , stem cells from human exfoliated deciduous teeth (SHEDs), periodontal ligament stem cells (PDLSCs), dental follicle precursor cells (DFPCs), alveolar bone-derived mesenchymal stem cells (ABMSCs), stem cells in the apical papilla of human immature permanent teeth , tooth germ progenitor cells (TGPCs), and gingiva-derived mesenchymal stem cells (GMSCs) . Multipong SHEDs . SHEDs png SHEDs . The expng SHEDs . Compareng SHEDs . SCAPs eng SHEDs . Moreoveng SHEDs . Culturein vitro  , an upstream regulatory molecule. It is critical for dentinogenesis and odontoblastic differentiation  mRNA expression in dental pulp cells (DPCs)  and Dspp is upregulated in DPCs exposed to Wnt3a (In vivo stem cell implantation assay suggested that the synergistic action of bone morphogenetic protein 9 (BMP9) and Wnt3a may enhance the odontoblastic differentiation of immortalized mouse stem cells of the apical papilla tissue of mouse lower incisor teeth (iSCAPs) , and dentin sialoprotein (DSP), leading to reduced canonical Wnt activity and inhibition of odontoblast maturation  . β-catens (DPCs) . In vitrto Wnt3a . In vivo(iSCAPs) . Wntlessntiation . Odontobturation . In vivoignaling . These rWnt genes are upregulated in DPSCs , in which Wnt/β-catenin pathway is inhibited by microRNA-328-3p (miR-328-3p)  short nucleolar RNA host gene 1 (-328-3p) .Wnt5a mutant mice, the expression of Bmp4 and Msh (muscle segment homeobox) Drosophila homolog 1 (Msx1) is attenuated in the dental mesenchyme, and delayed odontoblastic differentiation is observed  and BMP family intracellularly. Smads are divided into three categories: receptor-regulated Smads (R-Smads), including Smad1, Smad2, Smad3, Smad5, Smad8, and Smad9; common-partner Smads (co-Smads); Smad4, which is associated with phosphorylated R-Smads; and inhibitory Smads (I-Smads), including Smad6 and Smad7 .TGF family is multipotent in numerous biological processes . TGF-β sDSPP, RUNX2, and DMP1, which indicates that TGF-β1 positively induces odontoblastic differentiation of hDPSCs  homeobox gene 3 (Dlx3), Msx1, Msx2, paired box 9 (Pax9), DMP1, and DSPP , an Oriental phytomedicine, the phosphorylation of Smad1/5/8 is upregulated, and odontoblastic differentiation was enhanced via the Smad signaling pathway , and p38 protein kinases . ERK conThe p38 MAPK pathway is involved in odontoblastic differentiation. Activated p38a MAPK phosphorylates downstream TFs to initiate odontoblastic gene expression and hDPC differentiation, whereas blockage of p38α MAPK inhibits odontoblastic differentiation . In MDPC2) enhances DPSC differentiation by promoting the expression of p38, JNK, and ERK 2, as increased expression of Notch1, Notch2, Notch3, Delta1, Jagged1 and Hes1 was observed. This suggests that pulp capping with Ca(OH)2 promotes odontoblastic differentiation of DPCs  is observed in the tooth germ mesenchyme and increases during odontogenic differentiation in vivo and in vitro. When Zeb1 is inhibited, the differentiation of mDPCs is therefore reduced. Mechanistically, the expression of Runx2 and Dspp is promoted by ZEB1 respectively in the early and late phases of odontoblastic differentiation , overexpression of Klf5, Klf6, and Klf10 promotes odontoblastic differentiation. Mechanistically, the transcription of nd Klf10 . ATF6, and Klf10 . The expntiation . BTB andntiation . Hypoxiantiation . Moreoventiation .In the process of odontoblastic differentiation, posttranscriptional regulation is involved in odontoblastic differentiation. This review provides an overview of the regulation by microRNAs  and long noncoding RNAs (lncRNAs) at the posttranscriptional level.MiRNAs, a subgroup of endogenous ∼22-nucleotide-long noncoding RNA, have been implicated in posttranscriptional gene regulation . miRNAs miRNAs, including miR-338-3p, miR-21, miR-3065-5p, and miR-223-3p, can positively mediate odontoblastic differentiation, as shown in Dspp and Dmp1, indicating enhanced odontoblastic differentiation , and subsequently named it DANCR . The expRecent studies have identified other lncRNAs involved in odontoblastic differentiation. Tu et al. identified a novel lncRNA named CALB2, which enhances the odontoblastic differentiation of hDPCs via the CALB2/miR-30b-3p/RUNX2 axis by sponging miR-30b-3p and upregulating RUNX2 . The lncEpigenetic regulation, including DNA methylation and histone tail modification, has recently been shown to modulate odontoblastic differentiation, as shown in Klf4 and odontoblastic marker genes. SP1 modulates KLF4 via a demethylated binding site on a CpG island in KLF4 promoter region (6 A) methyltransferase METTL3 promotes odontoblastic differentiation. Absence of METTL3 in hDPCs inhibits NFIC translation. Consequently, the knockdown of METTL3 results in decreased odontogenic differentiation in vitro, and reduced dentin formation in the root has been observed in vivo (DNA methyltransferases (DNMTs), including DNMT1, DNMT3A, and DNMT3B, regulate DNA methylation, which tends to silence the promoter and enhancer classes . DNA metr region . N6-meth in vivo . These rEpigenetic modifications of histones include methylation, acetylation, phosphorylation, and ubiquitylation , among wHistone methyltransferases (HMTs) and demethylases (HDMs) control histone methylation, which is the epigenetic modulation critical in many psychological processes .HMTs are involved in odontogenesis. The four histone 3 lysine 9 (H3K9) MTs, G9a, GLP, PRDM2, and SUV39H1, were expressed abundantly in the tooth germ, and their expression reached a peak at E16.5 and E17.5. This suggests that the four H3K9MTs regulate tooth development and cell differentiation .Histone demethylase lysine(K)-specific demethylases (KDMs) tend to induce odontoblastic differentiation. KDM6B binds to the BMP2 promoter and activates odontogenic transcriptional genes by removing trimethylated H3K27 (H3K27me3). In DMSCs, knockdown and overexpression of KDM6B result in the attenuation and upregulation of odontoblastic differentiation, respectively . In SCAPEREG transcription is inhibited by the increased methylation of histone K4/36 in the EREG promoter. Therefore, FBXL11 inhibits odontoblastic differentiation of SCAPs  binds to the BCL6 co-repressor for activation; consequently, of SCAPs . KDM5A nof SCAPs . Inhibitof SCAPs .Histone acetyltransferases (HATs) and deacetylases (HDACs) are responsible for histone acetylation, which changes the interaction between histone proteins, DNA, and nuclear proteins, and serves as a type of epigenetic regulation during odontoblastic differentiation .DMP1, DSPP, DSP, OPN and OCN  chromatin remodeling complex. Knockdown of Baf45a leads to the downregulation of TFs that regulate odontoblast differentiation-related marker genes. It has been demonstrated that BAF45A induces remodeling of the promoters of genes that promote odontoblast differentiation in a transcriptional manner  is the most common etiologic mutation of TDO (TDO syndrome (Online Mendelian Human Genetics (OMIM) database 190320) is caused by n of TDO .Choi et al. developed transgenic mice expressing MT-DLX3 and observed evident dentin defects and enlarged unmineralized pulp in patients with TDO. MT-DLX3 has been demonstrated to affect odontoblastic differentiation, resulting in increased odontoblast apoptosis and distortion of dentin tubule production and dentin matrix formation, thereby downregulating dentin formation and taurodontism appeared .DLX3 (MU-DLX3) was reported to cause TDO syndrome  in syndrome . MU-DLX3syndrome . miR-675syndrome . Downregutations . MU-DLX3utations .TDO is an autosomal dominant (AD) condition characterized by anomalies in hair, teeth, and bones. TDO patients have kinky, curly hair which is featured and distinguished . PatientDental features including thin enamel, thin dentin, and taurodontism may be the most distinct characteristics of TDO patients , includiPatients with TDO mainly suffer from dental hypersensitivity, attrition, loss of tooth structure, dental abscesses, esthetic problems, and psychosocial problems . A comprRestorative treatments are required for patients with TDO to recover their tooth shape. Full crowns (prefabricated stainless steel crowns) are beneficial for TDO patients , as they307800), the most frequent form with a prevalence of 1:20000-60000, is a genetic disease characterized by defective mineralization of bones and tooth dentin, such as osteomalacia and radiolucent dentin (PHEX) on chromosome Xp22.1-22.2  was first described in 1989 as a syndrome characterized by lethal osteosclerotic bone dysplasia (FAM20C (NM_020223.3) as the cause of RS  and non-lethal (NLRS) (FAM20C have been identified in patients with LRS or NLRS (RS (OMIM ysplasia . It is aysplasia . In 2007se of RS . In 2009l (NLRS) . To date or NLRS .Fam20c , which encodes the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP) and leads to diminished ALP activity  is a rare systemic genetic disorder resulting from mutations in activity . Mutatiostations . Severe stations .ALPL gene mutations also lead to defective odontoblastic differentiation, as the canonical Wnt signaling pathway can be impaired by ALPL deficiency. In DPSCs from patients with HPP, there is a decrease in p-GSK3β and active β-catenin, and the expression levels of odontoblastic marker genes, including DSPP and DMP1, are attenuated. In normal DPSCs, downregulation and upregulation of ALPL inhibited and promoted the levels of p-GSK3β and active β-catenin, respectively. Therefore, the odontoblastic differentiation capacity of DPSCs was impaired (impaired .241510), infantile (OMIM 241500), perinatal (OMIM 146300), benign prenatal phosphatasia and pseudohypophosphatasia  was approved as a valid treatment for HPP patients . Dental 242900) and Elsahy-Waters syndrome  are discussed in this section, because their gene mutations may impair odontoblastic differentiation of DMSCs  (SMARCAL1 encodes a protein from the sucrose non-fermenting 2 (SNF2) family, which serves as a DNA annealing helicase involved in chromatin remodeling  . SMARCALmodeling , and SMAmodeling . In cultmodeling . It is cmodeling . Morimotd molars . In a cad molars . These cd molars .CDH11) , is an ultrarare AR syndrome caused by mutations in cadherin-11 (CDH11) . CDH11 mCDH11) . In contCDH11) . EWS is CDH11) . Dental CDH11) . In concSince the discovery of DPSCs in 2000 , the pas


Intratumoral injection of talimogene laherparepvec evokes a cytotoxic immune response. Therefore, the combination of talimogene laherparepvec with trabectedin and nivolumab may have synergistic effects in advanced sarcomas.2 q3 weeks), nivolumab intravenously (3 mg/kg q2 weeks), and intratumoral talimogene laherparepvec (1x108 plaque forming units/ml q2 weeks).This phase 2 trial was conducted from May 30, 2019 to January 31, 2022. Endpoints: Primary: Progression free survival rate at month 12. Secondary: Best overall response, progression free survival rate at 6 and 9 months, overall survival rate at 6, 9, and 12 months, incidence of conversion of an unresectable tumor to a resectable tumor, and incidence of adverse events. Eligible patients had to be ≥ 18 years of age, have advanced histologically proven sarcoma, at least 1 previous chemotherapy regimen, and at least one accessible tumor for intratumoral injection. Treatment: Trabectedin intravenously . Progression free survival rate at month 12, 36.7%. Confirmed Best Overall Response by Response Evaluation Criteria in Solid Tumors v1.1 = 3 partial responses, 30 stable disease, 6 progressive disease. Best Overall Response Rate, 7.7%, Disease Control Rate, 84.6%; median progression free survival, 7.8  months; 6-, 9-, 12-month progression free survival rates, 54.5%/45.9%/36.7%; median overall survival 19.3  months; 6-, 9- and 12-month overall survival rate, 86.9%/73.3%/73.3%. One patient had a complete surgical resection. Fifty percent of patients had a ≥ grade 3 treatment related adverse events which included anemia (6%), thrombocytopenia (6%), neutropenia (4%), increased alanine transaminase (4%), decreased left ventricular ejection fraction (4%), dehydration (4%), hyponatremia (4%).Taken together these data suggest that the TNT regimen is effective and safe for advanced previously treated sarcomas, and is worth being further studied in a randomized phase 3 trial as first- or second- line treatment for patients with advanced sarcomas. A starting dose of TVEC (1x106 PFU/ml) was initially given, followed 3 weeks later by a dose of 1x108 PFU/ml q2 weeks depending on tumor size three weeks later. Follow up computerized tomography (CT) or magnetic resonance imaging (MRI) were conducted within 30 days of enrollment and every 6 weeks thereafter. Per protocol, patients who did not complete one treatment cycle and had a follow up CT scan or MRI were replaced.This single center, open-label single-arm, phase 2 trial was conducted from May 30, 2019 to January 31, 2022. Treatment schedule: Trabectedin  and conducted in accordance with institutional and federal guidelines for human investigation in accordance with the Declaration of Helsinki. All participants were informed of the investigational nature of the study and provided written informed consent prior to enrollment. This trial was sponsored by Amgen. Ongoing safety oversight was conducted by the WIRB. Any serious unexpected adverse events were reported to the WIRB and the U.S. Food and Drug Administration.2.3via computerized tomography (CT) or magnetic resonance imaging (MRI), median PFS, PFS rate at 6 and 9 months, median OS, OS rate at 6, 9, and 12 months, incidence of conversion of an unresectable tumor to a resectable tumor, and incidence of adverse events related to trabectedin, nivolumab and talimogene laherparepvec. Exploratory endpoint was correlation of response with immune cell trafficking in the tumor microenvironment of resected tumors.The primary end point was PFS rate at month 12 per RECIST 1.1 . Seconda2.42.4.1Demographics, age at enrollment (<65 vs ≥65 and <75 vs ≥75 years), sex, subtypes of sarcoma, first- second- or third-line therapy, number of patients, patients with locally advanced or metastatic, resectable or unresectable tumors, and European Cooperative Oncology Group (ECOG) score were described using descriptive statistics.2.4.2The Intention-to Treat (ITT) population consisted of all subjects, who received at least one dose of trabectedin, nivolumab or talimogene laherparepvec. The modified Intention-to-Treat (mITT) population was composed of all patients who had completed the first cycle of trabectedin, nivolumab and talimogene laherparepvec and had a CT scan or MRI at the 6-week follow-up period. Patients who did not complete one treatment cycle and did not have a follow-up CT scan or MRI were replaced. The ITT population was used for analysis of overall survival and incidence of adverse events. The mITT population was used for analysis of the primary and secondary end points.2.4.3PFS at 12 months was assessed by RECIST v1.1  with the2.4.4Response was assessed utilizing RECISTv1.1 . Compute2.4.5Incidence and severity of adverse events and significant laboratory abnormalities were performed on all patients (ITT population). Patient incidence of all treatment related adverse events (TRAEs) were tabulated by system organ class and preferred term using National Cancer Institute Common Toxicity Criteria for Adverse Events Version 5 . Tables 2.4.6The analyses of the exploratory endpoints were descriptive in nature.2.4.7The number of patients studied were 39 evaluable patients in the mITT group. Patients who withdrew or did not complete the first 6-week cycle and first follow up CT scan/MRI were replaced. Although this study was not large enough to allow firm conclusions about safety or efficacy, it provided preliminary data on safety and efficacy that would be useful in planning future Phase 2 studies. A two-fold prolongation of median progression free survival in evaluable patients compared to median PFS reported with trabectedin alone . The cut-off point for PFS and OS was 1/31/2022. Eleven patients were not deemed evaluable for efficacy analysis because they did not complete the first treatment cycles and did not get a follow-up CT scan. Thirty-nine patients were evaluable for efficacy analysis.As shown in 3.3As of the Data Cut-off, 15 patients are known to be alive; of these, one patient (carcinosarcoma) is continuing nivolumab after completing one year of therapy and one patient (myoepithelioma) is continuing talimogene laherparepvec and nivolumab after completing one year of therapy. One patient with locally advanced rhabdomyosarcoma received intratumoral injections of talimogene laherparepvec, followed by surgical resection. She is in sustained remission 2 years s/p surgical resection on no further cancer therapy.As of data cut-off, 29 patients have died from disease progression, 6 patients withdrew due an adverse event and 6 patients were lost to follow-up. Supplementary materials are provided for all adverse events , and listings of deaths (C), patients who withdrew (D) and who were lost to follow up (E).3.43.5Safety was assessed by the incidence of treatment related and unrelated adverse events (AE) according to the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 .Twenty-five of 50 (50%) ITT patients had a = grade 3 TRAE. 3.6The correlation between response with immune cell trafficking in the tumor microenvironment of resected tumors: Stable disease was correlated with a significant decrease in tumor size by histopathologic examination in a patient with rhabdomyosarcoma  evaluated the efficacy and safety of pembrolizumab, a PD-1 inhibitor, in a two-cohort, single-arm, open-label Phase 2 multi-center study. The study showed favorable responses in patients with advanced STS and in patients whose molecular profile showed enhanced tumor infiltrating T cells and tumor associated macrophages expressing PD-L1 , 14, 25.In recent years, the TMEs of sarcoma histologic subtypes have been characterized , 25–28. There are a number of ways to exploit the immune system against sarcomas including immune checkpoint inhibitors, CAR-T cell therapy, natural killer cell therapy, virotherapy, dendritic cell therapy, and drugs that deplete the tumor microenvironment of M2 growth promoting macrophages , 25–31. Finally, the advent of sophisticated molecular profiling of tumors based on artificial intelligence and bioinformatics  is expecLimitations. The main limitations of this study are that it is a non-randomized single center study of a small number of patients with multiple subtypes of sarcoma including one patient with carcinosarcoma, an undifferentiated carcinoma. Indirect comparisons are not definitive statements about the superiority of a treatment regimen over another. The main strength of the study is that the results are promising and hypothesis generating which could be used for planning a Phase 2/3 study.5This non-randomized phase 2 study met its primary and secondary endpoints which demonstrates the efficacy and safety of the TNT regimen for advanced sarcomas. The treatment regimen is worth being further studied in a randomized phase 3 trial as first- or second- line treatment for patients with advanced sarcomas.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Western IRB. The patients/participants provided their written informed consent to participate in this study.Conception and Design: EG, SC, DB; Clinical Investigators: EG, SC, DQ, AM; Data Generation: EG, SC, WT, HC, CV, AA,VC-A; Data Analysis: EG, WT, SC, HC, NO, SM, AA, VC-A, DQ, DB, AM; Manuscript Writing and Approval: EG, WT, SC, HC, CV, AA, NO, SM, DB, VC-A, DQ, AM. All authors contributed to the article and approved the submitted version.


PTEN gene is an important and well-characterised tumour suppressor, known to be altered in many cancer types. Interestingly, the effect of the loss or mutation of PTEN is not dichotomous, and small changes in PTEN cellular levels can promote cancer development. Less well-known mechanisms regulating PTEN, with emerging importance, include the PTEN–miRNA–PTENP1 axis, which has been shown to play a critical role in the fine tuning of PTEN cellular levels. This mechanism, working at the post-transcriptional level, involves the interplay and competition between the PTEN transcript, its pseudogene long non-coding RNA transcripts, PTENP1, and microRNAs. Our growing knowledge of this mechanism has opened avenues for the development of strategies to alter the cellular levels of PTEN, miRNAs, and PTENP1 as a new frontier in cancer therapy.The PTEN) is a well characterised tumour suppressor, playing a critical role in the maintenance of fundamental cellular processes including cell proliferation, migration, metabolism, and survival. Subtle decreases in cellular levels of PTEN result in the development and progression of cancer, hence there is tight regulation of the expression, activity, and cellular half-life of PTEN at the transcriptional, post-transcriptional, and post-translational levels. PTENP1, the processed pseudogene of PTEN, is an important transcriptional and post-transcriptional regulator of PTEN. PTENP1 expression produces sense and antisense transcripts modulating PTEN expression, in conjunction with miRNAs. Due to the high sequence similarity between PTEN and the PTENP1 sense transcript, the transcripts possess common miRNA binding sites with the potential for PTENP1 to compete for the binding, or ‘sponging’, of miRNAs that would otherwise target the PTEN transcript. PTENP1 therefore acts as a competitive endogenous RNA (ceRNA), competing with PTEN for the binding of specific miRNAs to alter the abundance of PTEN. Transcription from the antisense strand produces two functionally independent isoforms (PTENP1-AS-α and PTENP1-AS-β), which can regulate PTEN transcription. In this review, we provide an overview of the post-transcriptional regulation of PTEN through interaction with its pseudogene, the cellular miRNA milieu and operation of the ceRNA network. Furthermore, its importance in maintaining cellular integrity and how disruption of this PTEN–miRNA–PTENP1 axis may lead to cancer but also provide novel therapeutic opportunities, is discussed. Precision targeting of PTENP1-miRNA mediated regulation of PTEN may present as a viable alternative therapy.The phosphatase and tensin homolog deleted on chromosome 10 ( PTEN), also known as mutated in multiple advanced cancers 1 (MMAC1) and TGFß-regulated and epithelial cell-enriched phosphatase 1 (TEP-1) [3 to PIP2, thereby negatively regulating the pro-proliferative and anti-apoptotic PI3K/Akt pathway to maintain cellular homeostasis [PTEN is frequently mutated and/or deleted in the inherited PTEN hamartoma tumour syndromes (PHTS) [The phosphatase and tensin homolog deleted on chromosome 10 ( (TEP-1) ,3, is a  (TEP-1) . The gen (TEP-1) ,4,5,6,7.eostasis ,9. The reostasis . PTEN iss (PHTS) ,13 and ms (PHTS) , skin (ms (PHTS) , and colPTEN with emerging importance include the PTEN–miRNA–PTENP1 axis, which has been shown to play a critical role in the fine tuning of PTEN regulation and cellular integrity. PTENP1 is a processed pseudogene of PTEN termed the phosphatase and tensin homolog pseudogene 1 , which is located on 9p13 (Gene ID: 101243555) [PTEN transcript; however, unlike PTEN, this transcript is not translated to produce a protein [PTENP1 are important in the modulation of PTEN expression at the transcriptional and post-transcriptional levels, respectively. The PTENP1 sense transcript (PTENP1-S), acting as a competitive endogenous RNA (ceRNA) of PTEN, leads to alterations in PTEN cellular abundance. The characteristics of this PTEN pseudogene lncRNA include similarities in their microRNA (miRNA) binding sites, and as such, PTENP1 can act as a decoy or ‘sponge’, competing for miRNAs that target PTEN. Disruption of the PTEN–miRNA–PTENP1 axis and ceRNA networks in carcinogenic progression is contemporary and is an exciting area in the discovery of regulatory mechanisms that are altered in cancer. In addition to its regulation of PTEN expression, PTENP1 is able to act as a tumour suppressor independent of its PTEN regulatory function as described in a recent review of the role of PTENP1 in human disorders with a focus on its tumour suppressor functionality [Less well-known regulatory mechanisms of 1243555) ,17,18. T protein . Althoug protein . The senionality .PTEN regulation in cancer development/progression through the well-known mechanisms of mutation, deletion, and alterations of PTEN structure and function, with a major focus on the role of the PTEN–miRNA–PTENP1 axis. The mechanisms of post-transcriptional regulation of PTEN, through interaction with its processed pseudogene (PTENP1) transcript (expressed as a lncRNA) and the cellular miRNA milieu, in the context of a cellular ceRNA network is discussed. Knowledge of the working of this regulatory mechanism will allow the identification of potential future novel therapeutic options. Precision targeting of the PTEN–miRNA–PTENP1 axis is important for the regulation of PTEN and may present as a viable alternative therapy to increase endogenous wild-type PTEN in tumours shown to have reduced PTEN levels.In this review, we outline the importance of PTEN is known to contribute to many cancers, highlighting the importance of this tumour suppressor in cancer initiation, progression, and metastasis. Germline mutations of PTEN are the cause of four autosomal dominant inherited syndromes: Cowden syndrome (CS) [PTEN germline mutations are reported in 25-85% of CS patients [PTEN mutations are also associated with a subset of patients with autistic behaviour and extreme macrocephaly [Germline and somatic mutation of ome (CS) , Bannayaome (CS) ,23, Protome (CS) , which some (CS) ,13. PTHSome (CS) ,13, thyrome (CS) ,13, renapatients ,25,26, 6patients ,22,25,27patients , and betpatients . Interesocephaly .PTEN are frequently associated with tumorigenesis with somatic alterations of PTEN being described in over 50% of cancers of various types [PTEN somatic mutations are most prevalent in prostate cancer [PTEN somatic alterations include the complete loss or inactivation of one allele  due to point mutations and/or deletions and/or epigenetic silencing through hypermethylation of the PTEN promoter, which is characteristic of some advanced and metastatic cancers [PTEN occurs at a lower incidence but is seen mostly in metastatic breast cancer, melanomas, and glioblastomas [Somatic mutations of us types . PTEN soe cancer , endomete cancer , melanome cancer ,34, non-e cancer ,36, kidne cancer , breast e cancer , and glie cancer . PTEN so cancers ,4. Deletlastomas ,4,40. Inlastomas . There alastomas ,42,43.PTEN is not dichotomous, and subtle changes in PTEN cellular levels have been shown to lead to deleterious consequences relating to tumour incidence, penetrance, and aggressiveness in several epithelial cancers [Pten mouse, it has been shown that in susceptible organs such as the prostate, PTEN protein expression levels need to reach dramatically low levels  to initiate tumorigenesis, however, in the mammary glands, a more subtle reduction  can initiate tumorigenesis [PTEN does not follow the ‘two-hit’ paradigm or stepwise model of tumour suppressor gene function but rather presents a new continuum model of tumorigenesis whereby tumorigenesis occurs in an incremental dose-dependent manner [PTEN expression was shown to gradually decrease with increasing gastric cancer progression [The effect of the loss or mutation of  cancers ,78. In tt manner ,78. Thisgression .PTEN expression occurs with hemizygous loss leading to tumour anti-cancer immune responses [PTEN expression has been shown in vivo, in mouse models, to be due to specific miRNAs. An example being PTEN as a target of mi-R22 in breast and prostate cancers, which have been shown to have a strong influence in a cancer immune TME, playing a role in cancer initiation, progression, and metastasis [There are several recent studies that have explored the relationship between PTEN loss and tumour immunity, showing PTEN loss contributes to alterations in the tumour microenvironment (TME) to produce an immunosuppressive niche. The evidence suggests that PI3K signalling may influence the composition and functionality of the TME, thereby modulating the immune response in cancer. Vidotto et al. (2023) analysed PTEN copy number in 9793 cases from 30 tumour types, derived from the Cancer Genome Atlas, and showed that reduced tumour esponses . In anotesponses . Overallesponses . There wesponses . Interesesponses ,84. Redutastasis . Importatastasis . These fPTEN is constitutively expressed in normal cells and, due to its critical role in several cellular processes, is closely regulated at the transcriptional, post-transcriptional. and post-translational levels to modulate expression, activity, and cellular half-life [PTEN regulation is the post-transcriptional regulation by its pseudogene long non-coding RNA (lncRNA) and microRNA (miRNA) [PTEN–miRNA–PTENP1 ceRNA network is discussed in detail in the sections below.alf-life . One of  (miRNA) ,88,89,90PTEN include, but are not limited to, miR-17, miR-19, miR-21, miR-26, and miR-214 [PTEN expression in bladder cancer [MicroRNAs are single-stranded RNAs comprised of 19-23 nucleotides. These small endogenous RNAs bind to complementary regions within the 3’ untranslated region (UTR) of their mRNA targets, whereby perfect complementarity leads to target degradation and imperfect complementarity leads to the suppression of translation ,88,89,90r cancer , invasivr cancer , renal cr cancer , gastricr cancer , gliomasr cancer , lung adr cancer , and in r cancer . Expressr cancer . Classifr cancer ,100,101.PTENP1 is a processed pseudogene of PTEN, transcription of which produces unique, bidirectional, sense and antisense, transcripts [PTENP1-S), which is a pseudogene lncRNA with high sequence similarity to the PTEN transcript. There is 97.8% sequence similarity within the corresponding coding region (CDS) (with only 18 mismatches) of the two transcripts [PTENP1-S transcript is approximately 1 kb shorter than that of the PTEN transcript. Overall, the 5’-UTR, the CDS, and the early sections of the 3’-UTR of PTEN and PTENP1–S share high sequence similarity (approximately 95%), however, the level of sequence similarity decreases drastically (approximately 50%) towards the later sections and end of the 3’-UTR [nscripts . Transcrnscripts ,19,103, e 3’-UTR .PTENP1-S transcript shares common miRNA binding sites with the PTEN mRNA, particularly at the start of the 3’-UTR, resulting in the ability of PTENP1-S and PTEN to compete for the binding of common miRNAs. It is now well known that PTENP1-S acts as a miRNA sponge to protect PTEN from translational repression in a competitive manner, resulting in a positive impact on PTEN expression levels. This competition involves the participation of the PTEN and PTENP1-S transcripts, as well as the miRNAs targeting these transcripts, in a ceRNA network. Experimentally validated miRNAs that have been shown to participate in the PTEN and PTENP1-S ceRNA network in various cancer types are summarised in PTEN and PTENP1-S in prostate cancer [Due to the high sequence similarity between the two transcripts, the e cancer , hepatoce cancer , clear ce cancer , and orae cancer . GainingPTENP1, transcription from the antisense strand produces two functionally independent isoforms, PTENP1-antisense-alpha (PTENP1-AS-α) and PTENP1-antisense-beta (PTENP1-AS-β) [cis overlap with the PTENP1-S transcript and the 5’-UTR of PTEN [PTENP1-AS-α isoform shares high sequence similarity with the 5’-UTR of PTEN and is most abundant in the nucleus [PTENP1-AS-α binds to the 5’-UTR of PTEN-associated transcripts, which allows for the localisation of PTENP1-AS-α to the PTEN promoter region and, in turn, recruits epigenetic modifiers, including chromatin remodelling proteins EZH2 and DNMT3A, which induce the H3K27me3 post-translational histone modification at the PTEN promoter, consequently leading to the negative transcriptional regulation of PTEN expression [PTENP1-AS-β transcript binds to the PTENP1-S transcript, which lacks a poly-A tail and provides stability to the PTENP1-S transcript through the formation of a PTENP1-S and PTENP1-AS-β complex that is exported into the cytoplasm, where PTENP1-S acts as a miRNA sponge to post-transcriptionally regulate PTEN through participation in the ceRNA network [In addition to the sense transcript of P1-AS-β) . Both is of PTEN ,117,118. nucleus ,117,118. network ,117,118 PTEN has been shown to be regulated by the PTENP1 sense transcript as PTENP1-S acts as a decoy to sequester miRNAs that would otherwise target and repress PTEN mRNA translation, thus maintaining or restoring PTEN protein levels [PTEN and PTENP1 ceRNA regulatory network.The endogenous competitive RNA (ceRNA) binding hypothesis, first postulated by Pandolfi and colleagues, states that endogenous RNAs, including mRNAs, transcribed pseudogenes, protein-coding genes, lncRNAs, and circular RNAs, compete to regulate each other through binding or sponging of shared miRNAs from the same cellular miRNA pool ,120. In n levels . This paPTEN and PTENP1 in a ceRNA network in cervical cancer [PTEN and PTENP1 functioning as ceRNAs in studies using CRISPR knockdown and silencing of PTEN and PTENP1 in DU145 prostate cancer cells [PTENP1 knockdown resulted in the repression of PTEN expression [PTENP1-AS alpha and beta isoforms resulted in the downregulation of both PTENP1 and PTEN [PTEN and PTENP1 by the anti-sense isoforms [PTEN and PTENP1 resulted in the repression of the PTENP1-AS transcripts, while the opposite effect was seen when PTENP1 was upregulated [PTEN and PTENP1 in a ceRNA network through the mutual regulation of each other’s expression levels [Despite the myriad of papers confirming the involvement of l cancer ,121, brel cancer ,111, gasl cancer ,122, oral cancer , clear cl cancer , and in l cancer , there hl cancer ,124. Sucer cells . PTENP1 pression . Additioand PTEN , confirmisoforms . Furtheregulated . This wan levels .PTEN is regulated by its first identified ceRNA, PTENP1, other ceRNAs in the form of lncRNAs and proteins that regulate PTEN, have since been identified [PTEN through ceRNA mechanisms, which include, but are not limited to, FER1L4 competing with PTEN for miR-106a [MEG interacts with PTEN in a ceRNA manner to bind to miR-19a in glioma [PTEN has been shown to be downregulated by the lncRNA HOTAIR through interaction with miR-29b in laryngeal squamous cell carcinoma [PTEN through ceRNA mechanisms include Linc-USP16, which competes for miR-21 in hepatocellular carcinoma, with PTEN expression increasing upon its overexpression [PTEN through a ceRNA regulatory mechanism as PTEN and CAS2 both possess miR-21 binding sites [PTEN through the ceRNA mechanism include LINC00702 [NEAT1 [RP11-79H23.3 [TP73-AS1 [ORLNC1 [PTEN ceRNA competitors, including TNRC6B, RB1, TP53, NRAS, KLF6, HIF1A, HIAT1, CTBP2, and TNKS2 [While entified . A numbemiR-106a  in gastrmiR-106a . The lncn glioma . PTEN haarcinoma . Additiopression ,131. CASng sites ,132,133.INC00702 , NEAT1 [2 [NEAT1 , RP11-79-79H23.3 , TP73-ASTP73-AS1 , and ORL [ORLNC1 . Additiond TNKS2 ,140,141;PTENP1 copy number loss and decreased PTENP1 expression have been reported in conjunction with PTEN loss and decreased expression in several cancer types as the result of either deletion or silencing due to promoter hypermethylation. The PTENP1 promoter has been shown to be hypermethylated in pancreatic adenocarcinoma, breast cancer, cervical cancer, ovarian cancer, and hepatocellular carcinoma cell lines [PTENP1 was found to be methylated in MDA-MB-231 cells but unmethylated in MCF-7 cells [PTENP1-S was methylated in endometrial cancer and hyperplasia but not in normal tissue [PTENP1 elevated PTENP1-S expression in normal endometrium tissue and endometrial hyperplasia from women aged 45 and over, and/or women approaching, or in, menopause [ll lines , as wellll lines , colorecll lines , clear cll lines ,143, andll lines . In brea-7 cells . PTENP1-l tissue . Interesenopause ,146  and true cellular levels are yet to be determined. In this context, the use of absolute quantitation methodologies would allow determination of the true cellular levels of PTEN, PTENP1-S, and PTENP1-AS transcripts in normal and cancer cells to help provide an understanding of the perturbations of these levels in cancer cells of various types. This information would be of great importance for our understanding of the contributions of these transcripts in cancer development and progression and would help form the basis of potential future transcript ratio altering therapies for cancer treatment.Generally, anscript . Additioanscript . A recenpression . Furthersurvival . To datePTENP1-S transcript is capable of functioning as a tumour suppressor independently of its PTEN regulatory effects and has been shown to have a growth suppressive role in numerous cancers including prostate [PTENP1 led to a decrease in cell proliferation, suppression of cell migration and invasion, and induction of apoptosis through downregulation of the AKT and MAPK signalling pathways as well as downregulation of critical cell cycle proteins cyclin A2 and CDK2, in breast cancer [PTENP1 expression levels as a potential future candidate prognostic biomarker due to its tumour suppressor activity. A systematic review and meta-analysis of the data from the literature has recently been published revealing the prognostic value of PTENP1 expression in cancer. Dai et al. indicate that low expression of PTENP1 might predict poor prognosis for various carcinomas [Interestingly, the prostate , gastricprostate , clear cprostate , gliomasprostate , hepatocprostate , bladderprostate ,112,154,prostate ,110,155,prostate , melanomprostate , and colprostate . In thest cancer . This furcinomas .PTENP1 has been shown to regulate PTEN outside the context of cancer. In smooth muscle cells, PTENP1 inhibits proliferation and enhances apoptosis [PTENP1 expression has also been shown to affect recovery by modulating the levels of miR-19b and miR-21 [PTENP1 expression has implications for fertility as it was shown to regulate the human endometrial epithelial adhesive capacity in vitro by regulating miR-590-3p, and PTENP1 was shown to be one of a set of highly expressed lncRNAs in human endometrial epithelial cells subjected to blastocyst conditioned medium [Additionally, poptosis . In spind miR-21 . Interesd medium .PTEN is shown in its evolutionary history and conservation. Tang et al. (2016) have reported the identification of 37 PTEN pseudogenes (PTENPs) in 65 mammalian genomes, predominantly in primates and rodents [PTENPs were shared among primates and rodents, others were shown to be species-specific. Of interest, these authors reported the presence of 17 copies of PTENPs in the naked mole rat, an anticancer model organism, with all genes sharing common miRNA binding sites with their PTEN counterpart [PTENP1 is well established, a recent report has shown regulation of PTENP1 by PTEN in glioblastoma cells [PTENP1 CpG island, Kovalenko et al. showed that DNA methylation increases PTENP1-S expression in the presence of WT-PTEN protein but decreases PTENP1-S expression if there is an absence of PTEN protein. They further demonstrated that the PTEN protein binds to the promoter region of PTENP1 and inhibits PTENP1-S expression if its CpG island is demethylated. Thus, in glioblastoma cells, PTENP1 is a downstream target of PTEN.The importance of this functional pseudogene and its regulation of  rodents . While snterpart . While rma cells . Using tPTENP1 3’-UTR resulted in sequestration of miRNAs, showing PTENP1 to be an important gene in the regulation of PTEN. Overexpression of the 3’-UTR of PTENP1 in cell lines and in vivo studies has been shown to upregulate PTEN, thereby blocking the PI3K/Akt pathway and decreasing cell proliferation and metastasis, and increasing apoptosis in prostate (DU145) [PTENP1 did not successfully restore PTEN to normal levels in head and neck squamous cell carcinoma cell lines, HN13 and HN30 [PTENP1 in oesophageal squamous cell carcinoma cells led to increased PTEN levels in Eca19 cells but not in TE-1 cells [PTENP1 was overexpressed in ER-positive breast cancer cells (MCF-7 and T-47D), PTEN expression decreased, and tumour growth was reported to be accelerated in MCF-7 cells [PTENP1 in ER-negative breast cancer cells (MDA-MB-231 and C3HBA), led to increased PTEN expression and inhibition of tumour progression [PTEN and PTENP1 expression in cells [PTEN-miR-200c-PTENP1 [PTEN and PTENP1, neglecting the multiplicity of miRNAs that are able to target and act as either tumour suppressors or oncomiRs, depending on the cell/tissue type in which they are present [PTEN–PTENP1 ceRNA network.Overexpression of the  (DU145) , renal ( (DU145) , liver ( (DU145) , breast  (DU145) ,111,157, (DU145) , gastric (DU145) , oesopha (DU145) , and end (DU145)  cancer cand HN30 . Additio-1 cells . In the -1 cells . When PT-7 cells . Contrasgression ,111,144.in cells . Therefoc-PTENP1 . All the present . An examPTEN mRNA levels have been shown to be restored in PTEN null cells both in vitro in prostate cancer cells and in vivo in mouse models of prostate cancer through the use of nanoparticles delivering PTEN mRNA [PTEN null cells resulted in inhibition of the PI3K–Akt pathway and also increased apoptosis. This work represents a new approach to PI3K–Akt pathway inhibition through the restoration of PTEN mRNA. Recently, PTEN mRNA was delivered via nanoparticles to restore PTEN levels and enhance anti-tumour immunity in melanoma and prostate cancer mouse models [The restoration of functional PTEN has been difficult; however, TEN mRNA . Additioe models . Furthere models .PTENP1 into hepatocellular carcinoma (HCC) cells resulted in increased PTENP1 levels in cells [PTENP1 expressing baculoviral vector into mice with HCC tumours also reduced tumour growth and cell proliferation, induced apoptosis and autophagy, and inhibited HCC cell properties [PTENP1 has been transferred from normal cells to bladder cancer cells, which resulted in a reduction in the progression of bladder cancer in vitro and in vivo [PTENP1 packaged into exosomes has also been transferred to U87MG glioblastoma cells to sponge miR-10a-5p and stabilise PTEN levels in a competitive manner [PTEN mRNA or PTENP1 in both in vitro and in vivo studies is a promising start for these candidates for future gene therapies in clinical trials  of specific PTEN-regulating miRNAs, it is tempting to consider these future therapies that may allow the fine tuning of PTEN cellular levels to achieve and maintain pre-cancerous levels. While manipulating the PTEN–miRNA–PTENP1 axis holds great promise for the future of cancer therapies, our knowledge of the mechanisms of post-transcriptional regulation of PTEN, the various competing components, and the complexities of their interactions needs further study to allow this to become a future reality. Notwithstanding this, future successful human trials delivering miRNAs and/or PTEN and PTENP1 transcripts have great potential in precision therapeutic targeting and the treatment of a broad range of PTEN-related malignancies.Alterations of the cellular levels of PTEN, miRNAs, and


ATP8B1 and ABCB11 genes, histopathology, and treatment response were examined retrospectively. Results: Seven Japanese patients with BRIC were enrolled . The median age at onset for BRIC-1 was 12 years; for BRIC-2, it was 1 month. Intermittent cholestatic attacks numbered from one to eight during the 11 years of median follow-up. Six patients received a mainstream education; only one patient attended special education. None developed cirrhosis. Three with BRIC-1 showed compound heterozygosity for a variant ATP8B1 gene, while one was heterozygous; two BRIC-2 patients showed compound heterozygosity in ABCB11 and one was heterozygous. Liver biopsy specimens obtained during cholestatic attacks showed fibrosis varying from none to moderate; inflammation was absent or mild. Rifampicin administered to three patients for cholestatic attacks was effective in all, as was cholestyramine in two of three. Conclusions: To our knowledge, this is the first East Asian multicenter study of BRIC patients. Onset age and number of cholestatic attacks varied. Rifampicin and cholestyramine were effective against attacks. No patient developed cirrhosis; most had normal growth and development. The long-term outcomes were satisfactory.Background: Few reports of benign recurrent intrahepatic cholestasis (BRIC) have focused on East Asian patients. We describe the clinicopathologic features, genetics, treatment, and outcomes in Japanese BRIC patients. Methods: We recruited patients with BRIC type 1 (BRIC-1) or 2 (BRIC-2) treated at four pediatric centers and one adult center between April 2007 and March 2022. Demographics, clinical course, laboratory results, molecular genetic findings concerning  ATP8B1) and ATP-Binding Cassette Subfamily B Member 11 (ABCB11) genes [Benign recurrent intrahepatic cholestasis (BRIC) is a rare autosomal recessive inherited liver disease associated with canalicular transport defects causing a mild form of progressive familial intrahepatic cholestasis (PFIC). First described by Summerskill and Walshe in 1959, BRIC is characterized by intermittent episodes of cholestasis without progression to liver failure ,3. While1) genes ,5. Sympt1) genes ,3. As BRWe conducted a multicenter, retrospective, observational study which complied with the ethical guidelines of the Declaration of Helsinki of 1975, as revised in 2013, and was approved by the Ethical Committee of Kurume University and its counterparts at other participating centers. This retrospective study was conducted using an opt-out method.ATP8B1 or ACBC11 gene, or alternatively two or more intermittent cholestatic attacks associated with at least a single allelic variant in the ATP8B1 or ACBC11 gene. Patients had no symptoms of cholestasis such as jaundice or pruritus except during a cholestatic attack. Demographic attributes, clinical course, laboratory blood tests, molecular genetic results concerning the ATP8B1 or ABCB11 gene, histopathologic findings in the liver, and details of treatment were ascertained retrospectively. Laboratory blood tests included serum ALT, GGT, total bilirubin (T Bil), D Bil, and TBA. Growth was evaluated according to height and weight, expressed in terms of standard deviation (SD) [Subjects were retrospectively enrolled patients with a clinical and genetic diagnosis of BRIC-1 or BRIC-2 who visited 4 pediatric centers and 1 adult center in Japan between April 2007 and March 2022. BRIC was defined by one or more intermittent cholestatic attacks (serum D Bil > 1.5 mg/dL) with symptoms such as jaundice or pruritus associated with biallelic variants in the ion (SD) . EfficacATP8B1 or ABCB11 genes detected via either targeted sequencing analysis or targeted next-generation sequencing using a previously reported custom-configured neonatal/infantile intrahepatic cholestasis gene panel including both genes [ATP8B1 and NM_003742.4 for ABCB11).We examined variants in patients’ th genes ,8,9,10. Liver biopsy specimens obtained from patients during a cholestatic attack were assessed pathologically based on the New Inuyama Classification of chronic hepatitis. In this classification, chronic hepatic disease is characterized according to the degree of fibrosis (F) as follows: F0 , F1 , F2 , F3 , or F4 . Additionally, the New Inuyama Classification assesses chronic hepatic disease activity (A) based on the degree of lymphocytic infiltration and necrosis of hepatocytes: A0 (no necro-inflammatory reaction), A1 (mild necro-inflammatory reaction), A2 (moderate necro-inflammatory reaction), and A3 (severe necro-inflammatory reaction) ,12. Immup values below 0.05 were considered to indicate statistical significance. Statistical analyses were carried out using GraphPad Prism , which was also used to produce related figures.Continuous variables are expressed as medians with minimum and maximum, while categorical variables are expressed as percentages. The Mann–Whitney U test was used where appropriate. Variables with 2-sided We enrolled seven Japanese BRIC patients . The characteristics of the patients are shown in ATP8B1 gene, while another had only one allele variant identified. Two patients with BRIC-2 had compound heterozygosity involving the ABCB11 gene, while another had only one allele variant identified. Seven pathogenic variants were identified in the ATP8B1 gene and five were detected in the ABCB11 gene. The ATP8B1 variants included two missenses, one nonsense, three deletions (frameshifts), and one splice-site change; the ABCB11 variants included three missenses and two nonsenses. The variants all differed among patients, with no high-frequency variants identified.The pathogenic gene variants in each patient are shown in p = 0.0682 and p = 0.0921, respectively; Jaundice and pruritus occurred in 100% of patients during attacks (7/7), while gray stool discoloration, fatigue, anorexia, and weight loss were noted in 57% (4/7), 29% (2/7), 29% (2/7), and 14% (1/7), respectively. No patient had any symptom between attacks. When we compared the serum liver function test results between cholestatic attacks and non-attack evaluations, serum T Bil peaked during attacks , as did D Bil  and TBA , while ALT  and GGT  ranged from normal to mildly elevated. Between cholestatic attacks, ALT , GGT , T Bil , D Bil , and TBA  showed nearly normal values. Serum T Bil, D Bil, and TBA showed significant differences between attack and non-attack periods, while attack-related changes in ALT and GGT fell short of significance , and one splice-site change, while the ABCB11 variants included three missenses and two nonsenses. Klomp et al. [Genetic testing showed that three patients among the four with BRIC-1 had compound heterozygosity for variants in the B11 gene ,5. In oup et al.  and van p et al. ,5. The dOur multicenter BRIC study included liver histopathologic findings in all patients. We found the degree of liver fibrosis to vary among patients from none to moderate, while histologic features of inflammation during cholestatic attacks were absent or mild. While most patients did not undergo a follow-up liver biopsy when cholestatic attacks had resolved, liver function blood test results, transient elastographic findings, and images including ultrasonography and computed tomography were normal at these times. No patients developed cirrhosis. In our results, intermittent cholestatic attacks in patients with BRIC were sometimes associated with mild to moderate fibrosis without progression to cirrhosis. However, careful long-term monitoring of patients with BRIC is needed, considering that some patients with a form of disease intermediate between BRIC and PFIC have experienced a delayed development of cirrhosis ,17,18.We also performed immunohistochemical examinations for BSEP and MRP2 in the liver biopsy specimens. BSEP participates in the transport of bile salts from hepatocytes to bile canaliculi, while MRP2 facilitates the elimination of conjugated bilirubin [Drugs and invasive measures have both been used to treat cholestatic episodes in patients with BRIC. Drugs include UDCA, phenobarbital, rifampicin, cholestyramine, antihistamines, prednisolone, and 4-phenylbutyrate, while invasive procedures include biliary drainage and plasmapheresis. Rifampicin has been reported to be the drug most effective against cholestatic attacks in patients with BRIC ,7. In ouAlthough our present study offers the strength of a multicenter study, a number of limitations are evident. First, relatively small numbers of BRIC subjects were enrolled because BRIC is a very rare disease in Japan. Second, we could not assess liver histopathology, including stage of fibrosis, in the absence of cholestasis because no follow-up liver biopsy was performed in most patients. Third, since our subjects were all Japanese, our findings might not be generalizable to patients in other East Asian countries or different ethnic groups. Prospective multicenter controlled studies including larger and more diverse populations might provide further information.In conclusion, we retrospectively clarified clinical, pathologic, and genetic features, treatment for cholestatic attacks, and long-term outcomes in Japanese patients with BRIC. To our knowledge, this is the first multicenter study of BRIC patients from East Asia. The age at onset and number of cholestatic attacks varied. Both rifampicin and cholestyramine were effective against cholestatic attacks. No patient developed cirrhosis, and most patients attained normal growth and development. The long-term outcomes among Japanese BRIC patients were satisfactory.


In the published article, there was an error regarding the affiliation for Seung-Tae Lee and Jong Rak Choi. As well as having affiliation 1, they should also have Dxome, Seoul, Republic of Korea.Materials and Methods, the manufacturer of our custom panel was missed.In the published article, there was an error. In Materials and Methods, “Genetic analysis and variant interpretation”, paragraph 1. This sentence previously stated:A correction has been made to “For the genetic diagnosis of IEI, we used a custom-designed clinical exome panel including 4,894 genes related to human genetic diseases, including IEI (Supplementary Table S1).”The corrected sentence appears below:“For the genetic diagnosis of IEI, we used a custom-designed clinical exome panel  including 4,894 genes related to human genetic diseases, including IEI (Supplementary Table S1).”