Autosomal recessive non-syndromic hearing loss genes in Pakistan during the previous three decades.

Hearing loss is a clinically and genetically heterogeneous disorder, with over 148 genes and 170 loci associated with its pathogenesis. The spectrum and frequency of causal variants vary across different genetic ancestries and are more prevalent in populations that practice consanguineous marriages. Pakistan has a rich history of autosomal recessive gene discovery related to non-syndromic hearing loss. Since the first linkage analysis with a Pakistani family that led to the mapping of the DFNB1 locus on chromosome 13, 51 genes associated with this disorder have been identified in this population. Among these, 13 of the most prevalent genes, namely CDH23, CIB2, CLDN14, GJB2, HGF, MARVELD2, MYO7A, MYO15A, MSRB3, OTOF, SLC26A4, TMC1 and TMPRSS3, account for more than half of all cases of profound hearing loss, while the prevalence of other genes is less than 2% individually. In this review, we discuss the most common autosomal recessive non-syndromic hearing loss genes in Pakistani individuals as well as the genetic mapping and sequencing approaches used to discover them. Furthermore, we identified enriched gene ontology terms and common pathways involved in these 51 autosomal recessive non-syndromic hearing loss genes to gain a better understanding of the underlying mechanisms. Establishing a molecular understanding of the disorder may aid in reducing its future prevalence by enabling timely diagnostics and genetic counselling, leading to more effective clinical management and treatments of hearing loss.

SARS-CoV-2 NSP12 associates with TRiC and the P323L substitution acts as a host adaption.

IMPORTANCE: SARS-CoV-2 has caused a worldwide health and economic crisis. During the course of the pandemic, genetic changes occurred in the virus, which have resulted in new properties of the virus-particularly around gains in transmission and the ability to partially evade either natural or vaccine-acquired immunity. Some of these viruses have been labeled Variants of Concern (VoCs). At the root of all VoCs are two mutations, one in the viral spike protein that has been very well characterized and the other in the virus polymerase (NSP12). This is the viral protein responsible for replicating the genome. We show that NSP12 associates with host cell proteins that act as a scaffold to facilitate the function of this protein. Furthermore, we found that different variants of NSP12 interact with host cell proteins in subtle and different ways, which affect function.

Heterogenous Gene Expression of Bicellular and Tricellular Tight Junction-Sealing Components in the Human Intestinal Tract.

A major site for the absorption of orally administered drugs is the intestinal tract, where the mucosal epithelium functions as a barrier separating the inside body from the outer environment. The intercellular spaces between adjacent epithelial cells are sealed by bicellular and tricellular tight junctions (TJs). Although one strategy for enhancing intestinal drug absorption is to modulate these TJs, comprehensive gene (mRNA) expression analysis of the TJs components has never been fully carried out in humans. In this study, we used human biopsy samples of normal-appearing mucosa showing no endoscopically visible inflammation collected from the duodenum, jejunum, ileum, colon, and rectum to examine the mRNA expression profiles of TJ components, including occludin and tricellulin and members of the claudin family, zonula occludens family, junctional adhesion molecule (JAM) family, and angulin family. Levels of claudin-3, -4, -7, -8, and -23 expression became more elevated in each segment along the intestinal tract from the upper segments to the lower segments, as did levels of angulin-1 and -2 expression. In contrast, expression of claudin-2 and -15 was decreased in the large intestine compared to the small intestine. Levels of occludin, tricellulin, and JAM-B and -C expression were unchanged throughout the intestine. Considering their segment specificity, claudin-8, claudin-15, and angulin-2 appear to be targets for the development of permeation enhancers in the rectum, small intestine, and large intestine, respectively. These data on heterogenous expression profiles of intestinal TJ components will be useful for the development of safe and efficient intestinal permeation enhancers.

A novel pathogenic variant in the MARVELD2 gene causes autosomal recessive non-syndromic hearing loss in an Iranian family.

BACKGROUND AND AIMS: Hearing loss (HL) is the most common sensorineural disorder and one of the most common human defects. HL can be classified according to main criteria, including: the site (conductive, sensorineural and mixed), onset (pre-lingual and post-lingual), accompanying signs and symptoms (syndromic and non-syndromic), severity (mild, moderate, severe and profound) and mode of inheritance (Autosomal recessive, autosomal dominant, X-linked and mitochondrial). Autosomal recessive non-syndromic HL (ARNSHL) forms constitute a major share of the HL cases. In the present study, next-generation sequencing (NGS) was applied to investigate the underlying etiology of HL in a multiplex ARNSHL family from Khuzestan province, southwest Iran. METHODS: In this descriptive study, 20 multiplex ARNSHL families from Khuzestan province, southwest of Iran were recruited. After DNA extraction, genetic linkage analysis (GLA) was applied to screen for a panel of more prevalent loci. One family, which was not linked to these loci, was subjected to Otogenetics deafness Next Generation Sequencing (NGS) panel. RESULTS: NGS results showed a novel deletion-insertion variant (c.1555delinsAA) in the MARVELD2 gene. The variant which is a frameshift in the seventh exon of the MARVELD2 gene fulfills the criteria of being categorized as pathogenic according to the American College of Medical Genetics and Genomics (ACMG) guideline. CONCLUSION: NGS is very promising to identify the molecular etiology of highly heterogeneous diseases such as HL. MARVELD2 might be important in the etiology of HL in this region of Iran.

Altered Structural Expression and Enzymatic Activity Parameters in Quiescent Ulcerative Colitis: Are These Potential Normalization Criteria?

Mucosal healing determined by endoscopy is currently the remission standard for ulcerative colitis (UC). However, new criteria for remission are emerging, such as histologic normalization, which appears to correlate better to the risk of relapse. Here, we study mucosal healing on a molecular and functional level in quiescent UC. We obtained endoscopic biopsies from 33 quiescent UC patients and from 17 controls. Histology was assessed using Geboes score. Protein and mRNA levels were evaluated for the tight junction proteins claudin-2, claudin-4, occludin, and tricellulin, as well as Cl-/HCO3- exchanger DRA, and cyclo-oxygenase enzymes (COX-1, COX-2). The mucosal activity of COX-1 and COX-2 enzymes was assessed in modified Ussing chambers, measuring electrogenic ion transport (short-circuit current, SCC). Chronic inflammation was present in most UC patients. The protein level of claudin-4 was reduced, while mRNA-levels of claudin-2 and claudin-4 were upregulated in UC patients. Surprisingly, the mRNA level of COX-1 was downregulated, but was unaltered for COX-2. Basal ion transport was not affected, while COX-2 inhibition induced a two-fold larger decrease in SCC in UC patients. Despite being in clinical and endoscopic remission, quiescent UC patients demonstrated abnormal mucosal barrier properties at the molecular and functional level. Further exploration of mucosal molecular signature for revision of current remission standards should be considered.

Tricellulin Effect on Paracellular Water Transport.

In epithelia, large amounts of water pass by transcellular and paracellular pathways, driven by the osmotic gradient built up by the movement of solutes. The transcellular pathway has been molecularly characterized by the discovery of aquaporin membrane channels. Unlike this, the existence of a paracellular pathway for water through the tight junctions (TJ) was discussed controversially for many years until two molecular components of paracellular water transport, claudin-2 and claudin-15, were identified. A main protein of the tricellular TJ (tTJ), tricellulin, was shown to be downregulated in ulcerative colitis leading to increased permeability to macromolecules. Whether or not tricellulin also regulates water transport is unknown yet. To this end, an epithelial cell line featuring properties of a tight epithelium, Madin-Darby canine kidney cells clone 7 (MDCK C7), was stably transfected with small hairpin RNA (shRNA) targeting tricellulin, a protein of the tTJ essential for the barrier against passage of solutes up to 10 kDa. Water flux was induced by osmotic gradients using mannitol or 4 and 40 kDa-dextran. Water flux in tricellulin knockdown (KD) cells was higher compared to that of vector controls, indicating a direct role of tricellulin in regulating water permeability in a tight epithelial cell line. We conclude that tricellulin increases water permeability at reduced expression.

Apoptotic Fragmentation of Tricellulin.

Apoptotic extrusion of cells from epithelial cell layers is of central importance for epithelial homeostasis. As a prerequisite cell-cell contacts between apoptotic cells and their neighbors have to be dissociated. Tricellular tight junctions (tTJs) represent specialized structures that seal polarized epithelial cells at sites where three cells meet and are characterized by the specific expression of tricellulin and angulins. Here, we specifically addressed the fate of tricellulin in apoptotic cells. METHODS: Apoptosis was induced by staurosporine or camptothecin in MDCKII and RT-112 cells. The fate of tricellulin was analyzed by Western blotting and immunofluorescence microscopy. Caspase activity was inhibited by Z-VAD-FMK or Z-DEVD-FMK. RESULTS: Induction of apoptosis induces the degradation of tricellulin with time. Aspartate residues 487 and 441 were identified as caspase cleavage-sites in the C-terminal coiled-coil domain of human tricellulin. Fragmentation of tricellulin was inhibited in the presence of caspase inhibitors or when Asp487 or Asp441 were mutated to asparagine. Deletion of the tricellulin C-terminal amino acids prevented binding to lipolysis-stimulated lipoprotein receptor (LSR)/angulin-1 and thus should impair specific localization of tricellulin to tTJs. CONCLUSIONS: Tricellulin is a substrate of caspases and its cleavage in consequence contributes to the dissolution of tTJs during apoptosis.

Molecular genetics of MARVELD2 and clinical phenotype in Pakistani and Slovak families segregating DFNB49 hearing loss.

Pathogenic mutations of MARVELD2, encoding tricellulin, a tricelluar tight junction protein, cause autosomal recessive non-syndromic hearing loss (DFNB49) in families of Pakistan and Czech Roma origin. In fact, they are a significant cause of prelingual hearing loss in the Czech Roma, second only to GJB2 variants. Previously, we reported that mice homozygous for p.Arg497* variant of Marveld2 had a broad phenotypic spectrum, where defects were observed in the inner ear, heart, mandibular salivary gland, thyroid gland and olfactory epithelium. The current study describes the types and frequencies of MARVELD2 alleles and clinically reexamines members of DFNB49 families. We found that MARVELD2 variants are responsible for about 1.5 % (95 % CI 0.8-2.6) of non-syndromic hearing loss in our cohort of 800 Pakistani families. The c.1331+2T>C allele is recurrent. In addition, we identified a novel large deletion in a single family, which appears to have resulted from non-allelic homologous recombination between two similar Alu short interspersed elements. Finally, we observed no other clinical manifestations co-segregating with hearing loss in DFNB49 human families, and hypothesize that the additional abnormalities in the Marveld2 mutant mouse indicates a critical non-redundant function for tricellulin in other organ systems.

Analysis of the 'angulin' proteins LSR, ILDR1 and ILDR2--tricellulin recruitment, epithelial barrier function and implication in deafness pathogenesis.

Tricellular tight junctions (tTJs) seal the extracellular space at tricellular contacts (TCs), where the corners of three epithelial cells meet. To date, the transmembrane proteins tricellulin and lipolysis-stimulated lipoprotein receptor (LSR) are known to be molecular components of tTJs. LSR recruits tricellulin to tTJs, and both proteins are required for the full barrier function of epithelial cellular sheets. In the present study, we show that two LSR-related proteins, immunoglobulin-like domain-containing receptor (ILDR) 1 and ILDR2, are also localized at TCs and recruit tricellulin. At least one of LSR, ILDR1 and ILDR2 was expressed in most of the epithelial tissues in mice. The expressions of LSR, ILDR1 and ILDR2 were generally complementary to each other, although LSR and ILDR1 were co-expressed in some epithelia. ILDR1 was required for the establishment of a strong barrier of the epithelium, similar to LSR, when introduced into cultured epithelial cells, whereas ILDR2 provided a much weaker barrier. We further analyzed human ILDR1, mutations in which cause a familial deafness, DFNB42, and found that most DFNB42-associated ILDR1 mutant proteins were defective in recruitment of tricellulin. We also found that tricellulin mutant proteins associated with another familial deafness, DFNB49, were not recruited to TCs by ILDR1. These findings show the heterogeneity of the molecular organization of tTJs in terms of the content of LSR, ILDR1 or ILDR2, and suggest that ILDR1-mediated recruitment of tricellulin to TCs is required for hearing. Given their common localization at epithelial cell corners and recruitment of tricellulin, we propose to designate LSR, ILDR1 and ILDR2 as angulin family proteins.

Increased permeability of the epithelium of middle ear cholesteatoma.

OBJECTIVE: We investigated the electrical impedance of and the expressions of tight junction molecules in the cholesteatoma epithelium to provide supporting evidence for the acid lysis theory of bone resorption in middle ear cholesteatoma. METHODS: Study subjects were patients with primary acquired middle ear cholesteatoma and those with non-cholesteatomatous chronic otitis media who underwent tympanomastoidectomy. The electrical impedance of the cholesteatoma epithelium was measured during tympanomastoidectomy by loading alternating currents of 320 Hz and 30.7 kHz. The expressions of tricellulin (MARVELD2), claudin-1 (CLDN1) and claudin-3 (CLDN3) were examined by fluorescence immunohistochemistry and quantitative reverse transcription-polymerase chain reaction. RESULTS: The electrical impedance of the cholesteatoma epithelium was significantly lower than that of the post-auricular skin and external auditory canal skin at both 320 Hz and 30.7 kHz. Immunoreactivity for MARVELD2, CLDN1 and CLDN3 was localised mainly in the granular layer, and to lesser degree, in the horny and spinous layers in both the cholesteatoma tissue and post-auricular skin. Fluorescence intensity was moderate for MARVELD2, weak for CLDN1 and strong for CLDN3. The expressions of MARVELD2, CLDN1 and CLDN3 mRNA were significantly lower in the cholesteatoma tissue than in the post-auricular skin. CONCLUSIONS: These results indicate the increased permeability of the cholesteatoma epithelium and suggest that this change is, at least partially, dependent on the decrease in the expressions of the tight junction molecules. This evidence supports the acid lysis hypothesis of bone resorption in cholesteatoma.

Novel compound heterozygous variants in MARVELD2 causing autosomal recessive hearing loss in two Chinese families.

BACKGROUND: Hereditary hearing loss is an important component of congenital hearing loss. MARVELD2 (OMIM ID:610572), located in the DFNB49 locus, which encodes a tight junction protein tricellulin playing an important role in the sensory epithelial barrier of the inner ear, may contribute to nonsyndromic autosomal recessive hereditary hearing loss. METHODS: Two Han Chinese pedigrees with hearing loss underwent clinical and genetic analyses. Variants were detected by targeted next-generation sequencing and sequencing data were compared with the Human Genome Reference (GRCh 37/hg 19) to identify mutant genes and loci. Furthermore, online tools such as RDDC, SpliceAI, and REVEL were used to predict risks from different variants. RESULTS: Both two probands failed neonatal hearing screening and were diagnosed with sensorineural hearing loss. A total of 3 mutations were detected in the two families, c.1331+1G>A, c.1325A>G, and c.782G>A. According to ACMG/AMP guidelines, they were judged to be pathogenic, uncertain significance, and uncertain significance, respectively. CONCLUSIONS: These findings contribute to a better understanding of the relationship between different variants of MARVELD2 and hearing. This could further expand the spectrum of deafness gene mutations and contribute to deafness patient management and genetic counseling.

Mfsd2a regulates the blood-labyrinth-barrier formation and function through tight junctions and transcytosis.

The Blood-Labyrinth Barrier (BLB) is pivotal for the maintenance of lymphatic homeostasis within the inner ear, yet the intricacies of its development and function are inadequately understood. The present investigation delves into the contribution of the Mfsd2a molecule, integral to the structural and functional integrity of the Blood-Brain Barrier (BBB), to the ontogeny and sustenance of the BLB. Our empirical findings delineate that the maturation of the BLB in murine models is not realized until approximately two weeks post-birth, with preceding stages characterized by notable permeability. Transcriptomic analysis elucidates a marked augmentation in Mfsd2a expression within the lateral wall of the cochlea in specimens exhibiting an intact BLB. Moreover, both in vitro and in vivo assays substantiate that a diminution in Mfsd2a expression detrimentally impacts BLB permeability and structural integrity, principally via the attenuation of tight junction protein expression and the enhancement of endothelial cell transcytosis. These insights underscore the indispensable role of Mfsd2a in ensuring BLB integrity and propose it as a viable target for therapeutic interventions aimed at the amelioration of hearing loss.

Zinc supplementation modifies tight junctions and alters barrier function of CACO-2 human intestinal epithelial layers.

BACKGROUND: Zinc deficiency is known to result in epithelial barrier leak in the GI tract. Precise effects of zinc on epithelial tight junctions (TJs) are only beginning to be described and understood. Along with nutritional regimens like methionine-restriction and compounds such as berberine, quercetin, indole, glutamine and rapamycin, zinc has the potential to function as a TJ modifier and selective enhancer of epithelial barrier function. AIMS: The purpose of this study was to determine the effects of zinc-supplementation on the TJs of a well-studied in vitro GI model, CACO-2 cells. METHODS: Barrier function was assessed electrophysiologically by measuring transepithelial electrical resistance (Rt), and radiochemically, by measuring transepithelial (paracellular) diffusion of 14C-D-mannitol and 14C-polyethyleneglycol. TJ composition was studied by Western immunoblot analyses of occludin, tricellulin and claudins-1 to -5 and -7. RESULTS: Fifty- and 100-µM zinc concentrations (control medium is 2 µM) significantly increase Rt but simultaneously increase paracellular leak to D-mannitol. Claudins 2 and 7 are downregulated in total cell lysates, while occludin, tricellulin and claudins-1, -3, -4 and -5 are unchanged. Claudins-2 and -7 as well as tricellulin exhibit decreased cytosolic content as a result of zinc supplementation. CONCLUSIONS: Zinc alters CACO-2 TJ composition and modifies TJ barrier function selectively. Zinc is one of a growing number of "nutraceutical" substances capable of enhancing epithelial barrier function, and may find use in countering TJ leakiness induced in various disease states.

Different expressions of erbB1/2 and tight junction proteins in hypertrophic inferior turbinates and nasal polyps.

Both inferior turbinate hypertrophy and nasal polyp formation entail the enlargement of the nasal mucosa caused by rhinosinusitis, but their macro/microscopic and clinical findings differ markedly. This study aimed at investigating differences in the expressions of erbB1/2 and the tight junction proteins, claudin-1 and tricellulin, in the two tissues. Ten inferior turbinates and ten nasal polyps were collected. The expressions of erbB1/2, claudin-1, and tricellulin were examined by fluorescence immunohistochemistry and by quantitative real-time transcription-polymerase chain reaction (qRT-PCR). The eosinophil count and % of nasal gland area in the mucosa were also measured. The fluorescence intensities in the inferior turbinates were higher for erbB1/2 and lower for claudin-1 than those in the nasal polyps. The results of qRT-PCR were consistent with the immunohistochemical findings for erbB1/2. The quantity of tricellulin mRNA was significantly higher in the inferior turbinates than in the nasal polyps. The % of nasal gland area was significantly higher but the eosinophil count was significantly lower in the inferior turbinate than in the nasal polyp. These results suggest that the underlying pathogenesis of hypertrophic inferior turbinates and nasal polyps is likely to differ with respect to regeneration/proliferation and thus the remodeling process.

Epidemiology, etiology, genetic variants in non- syndromic hearing loss in Iran: A systematic review and meta-analysis.

OBJECTIVES: Hearing loss is one of the most common heterogeneous complicated disorders worldwide. We previously analyzed the results of published data on non-syndromic hearing loss in the Iranian population systematically. A broad range of genes is a challenge for molecular screening and clinical diagnosis in our populations on the ground of distinct genetics. The aim of this study was to analyze the role and frequency of the variants accountable for non-syndromic hearing loss (NSHL) in the Iranian population. These were identified with different methods including whole exome sequencing (WES), next-generation sequencing (NGS), targeted genomic enrichment and massively parallel sequencing (TGE + MPS), autozygosity mapping, STR markers, linkage analysis, and direct sequencing. This is the comprehensively study focusing on classifying 13 common NSHL genes according to their frequencies. Previous studies have not studied different regions and the Iranian population, and this is the definitive study on the topic. METHODS: We searched Scopus, PubMed, Science Direct databases, and Google Scholar. After a systematic review of the evidence 95 studies were considered then 31 studies were eligible for meta-analysis. In total, 6995 families, 358 variants, and 117 novel variants were included. Statistical analyses were conducted using Stata SE version 11 software. The inverse variance method enjoyed combining data. Heterogeneity of the preliminary results was assessed using Q (Cochrane test), and I square index. Random effects or fixed models were applied to combine the results, relying on the degree of heterogeneity. Point and pooled prevalence of variants acting on different regions were illustrated by forest plots. RESULTS: The total prevalence of at least one variant of GJB2 and SLC26A genes was estimated at 26% and 5%, respectively. Variant c.35delG accounted for 18% of the GJB2 variants while 1% of these variants were novel ones. The next most common variants in the GJB2 gene were c.109G>A at 3.5% and c.-23+1G>A at 2.3%. Moreover, the prevalence of GJB2 gene variants varied on average 0.002% from one region to another in Iran (p=0.849). Our meta-analysis also showed that the frequency of at least one variant of MYO15A varied between 1.2% and 12.5%. Corresponding prevalences for the other variants were as follows: ILDR1 (3.5%-3.7%), CDH23 (2%-10%), PJVK (1.4%-33%), TECTA (1.3%-6.7%), MYO6 (2%-4.8%), TMC1 (1.8%-2%), MYO7A (0.7%-5%), MARVELD2 (0.7-5%), OTOF (0.7%-4%), LRTOMT (0.7%-2.5%). Finally, we did not find any relationship between geographic area and the presence of these variants. CONCLUSION: GJB2 gene variants were the most common cause of NSHL in Iran. Understanding the prevalence of NSHL gene frequency in Iran may be the foundation for future studies in an Iranian population which may lead to future NSHL therapy.

DFNB49 is an important cause of non-syndromic deafness in Czech Roma patients but not in the general Czech population.

Due to endogamy, the Roma have a higher risk for autosomal recessive (AR) disorders. We used homozygosity mapping on single-nucleotide polymorphism chips in one Czech Roma consanguineous family with non-syndromic hearing loss (NSHL). The second largest homozygous region in a deaf patient was mapped to the previously reported DFNB49 region. The MARVELD2 gene was recently reported as a causal gene for NSHL DFNB49. Sequencing of the MARVELD2 gene revealed a previously reported homozygous mutation c.1331+2 T>C (IVS4 + 2 T>C) in the deaf child. Subsequently, the same mutation was found in two more Roma families from an additional 19 unrelated Czech Roma patients with deafness tested for the MARVELD2 gene. To explore the importance of MARVELD2 mutations and DFNB49 for the general Czech and Central European population with early hearing loss we also tested 40 unrelated Czech patients with AR NSHL. No pathogenic mutation in the MARVELD2 gene was found in a group of 40 Czech non-Roma patients. Mutations in the MARVELD2 gene seem to be a significant cause of early NSHL in Czech Roma and this gene should be tested in this group of patients after GJB2.

Exome sequencing of families from Ghana reveals known and candidate hearing impairment genes.

We investigated hearing impairment (HI) in 51 families from Ghana with at least two affected members that were negative for GJB2 pathogenic variants. DNA samples from 184 family members underwent whole-exome sequencing (WES). Variants were found in 14 known non-syndromic HI (NSHI) genes [26/51 (51.0%) families], five genes that can underlie either syndromic HI or NSHI [13/51 (25.5%)], and one syndromic HI gene [1/51 (2.0%)]. Variants in CDH23 and MYO15A contributed the most to HI [31.4% (16/51 families)]. For DSPP, an autosomal recessive mode of inheritance was detected. Post-lingual expression was observed for a family segregating a MARVELD2 variant. To our knowledge, seven novel candidate HI genes were identified (13.7%), with six associated with NSHI (INPP4B, CCDC141, MYO19, DNAH11, POTEI, and SOX9); and one (PAX8) with Waardenburg syndrome. MYO19 and DNAH11 were replicated in unrelated Ghanaian probands. Six of the novel genes were expressed in mouse inner ear. It is known that Pax8-/- mice do not respond to sound, and depletion of Sox9 resulted in defective vestibular structures and abnormal utricle development. Most variants (48/60; 80.0%) have not previously been associated with HI. Identifying seven candidate genes in this study emphasizes the potential of novel HI genes discovery in Africa.

Angulin-1 seals tricellular contacts independently of tricellulin and claudins.

Tricellular tight junctions (tTJs) are specialized tight junctions (TJs) that seal the intercellular space at tricellular contacts (TCs), where the vertices of three epithelial cells meet. Tricellulin and angulin family membrane proteins are known constituents of tTJs, but the molecular mechanism of tTJ formation remains elusive. Here, we investigated the roles of angulin-1 and tricellulin in tTJ formation in MDCK II cells by genome editing. Angulin-1-deficient cells lost the plasma membrane contact at TCs with impaired epithelial barrier function. The C terminus of angulin-1 bound to the TJ scaffold protein ZO-1, and disruption of their interaction influenced the localization of claudins at TCs, but not the tricellular sealing. Strikingly, the plasma membrane contact at TCs was formed in tricellulin- or claudin-deficient cells. These findings demonstrate that angulin-1 is responsible for the plasma membrane seal at TCs independently of tricellulin and claudins.

Association of tricellulin expression with poor colorectal cancer prognosis and metastasis.

Tricellulin is a tight­junction transmembrane protein that regulates cell­cell interactions. Altered tricellulin expression could promote tumor cell invasions and metastasis in human cancers. The present study assessed tricellulin expression in colorectal cancer tissues for any association with clinicopathological features of colorectal cancer patients and then investigated the underlying molecular events using quantitative proteomic analysis and in vitro experiments. Tissue samples from 98 colorectal cancer patients and 15 volunteers were collected for immunohistochemistry. Colorectal cell lines were used to overexpress or knockdown tricellulin expression in various assays. The data revealed that upregulated tricellulin expression was associated with lymph node and distant metastases and poor prognosis, while tricellulin overexpression promoted colorectal cancer cell migration and invasion in vitro. In contrast, tricellulin knockdown had positive effects on the tumor cells. Furthermore, TMT­LC­MS/MS and bioinformatics analyses revealed that tricellulin was involved in EMT and reduction of apoptosis through the NF­κB signaling pathway. These findings highlight for the first time the significance of tricellulin in colorectal cancer development and progression. Further study may validate tricellulin as a novel biomarker and target for colorectal cancer.