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.

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 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.

Along with initiatives to understand the pathophysiology of stroke in detail and to identify neuroprotective targets, cell-stabilizing elements have gained increasing attention. Although cell culture experiments have indicated that tricellulin, α-catenin and microfibrillar-associated protein 5 (MFAP5) contribute to cellular integrity, these elements have not yet been investigated in the ischemic brain. Applying immunofluorescence labeling, this study explored tricellulin, MFAP5 and α-catenin in non-ischemic and ischemic brain areas of mice (24, 4 h of ischemia) and rats (4 h of ischemia), along with collagen IV and fibronectin as vascular and extracellular matrix constituents and microtubule-associated protein 2 (MAP2) and neurofilament light chain (NF-L) as cytoskeletal elements. Immunosignals of tricellulin and notably MFAP5 partially appeared in a fiber-like pattern, and α-catenin appeared more in a dotted pattern. Regional associations with vascular and extracellular constituents were found for tricellulin and α-catenin, particularly in ischemic areas. Due to ischemia, signals of tricellulin, MFAP5 and α-catenin decreased concomitantly with MAP2 and NF-L, whereby MFAP5 provided the most sensitive reaction. For the first time, this study demonstrated ischemia-related alterations in tricellulin, MFAP5 and α-catenin along with the vasculature, extracellular matrix and cytoskeleton. Confirmatory studies are needed, also exploring their role in cellular integrity and the potential for neuroprotective approaches in stroke.

The underlying pathophysiology of irritable bowel syndrome (IBS) is still unclear. Our aim was to investigate the pathophysiological mechanisms of diarrhea, constipation, and antigen uptake in mixed-type IBS (IBS-M).
Colonoscopic biopsies were obtained from IBS-M patients. Epithelial transport and barrier function of colonic mucosae were characterized in Ussing chambers using impedance spectroscopy. Mucosal permeability to macromolecules was measured. Western blotting for tight junction (TJ) proteins was performed and their subcellular localization was visualized by confocal microscopy. RNA-sequencing was performed for gene expression and signaling pathway analysis.
In IBS-M, epithelial resistance and ENaC-dependent sodium absorption were unchanged, while short-circuit current reflecting chloride secretion was reduced. Concomitantly, epithelial permeability for fluorescein and FITC-dextran-4000 increased. TJ protein expression of occludin decreased, whereas claudins were unaltered. Confocal microscopy revealed the de-localization of tricellulin from tricellular TJs. Involved pathways were detected as proinflammatory cytokine pathways, LPS, PGE2, NGF, and vitamin D.
Decreased anion secretion explains constipation in IBS-M, while ion permeability and sodium absorption were unaltered. Reduced occludin expression resulted in the delocalization of tricellulin from the tricellular TJ, leading to increased macromolecular permeability that contributes to antigen influx into the mucosa and perpetuates a low-grade inflammatory process.

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.

Tricellular junctions play a critical role in regulating epithelial barrier function. In this issue, Cho et al. (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202009037) demonstrate a novel interaction between tricellulin and α-catenin, which connects tricellular junctions to the actomyosin cytoskeleton, thus supporting the epithelial barrier at cell vertices.

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.

Tight junctions (TJs) are composed of a claudin-based anastomosing network of TJ strands at which plasma membranes of adjacent epithelial cells are closely attached to regulate the paracellular permeability. Although the TJ proteins occludin and tricellulin have been known to be incorporated in the TJ strand network, their molecular functions remain unknown. Here, we established tricellulin/occludin-double knockout (dKO) MDCK II cells using a genome editing technique and evaluated the structure and barrier function of these cells. In freeze-fracture replica electron microscopy, the TJ strands of tricellulin/occludin-dKO cells had fewer branches and were less anastomosed compared with the controls. The paracellular permeability of ions and small tracers was increased in the dKO cells. A single KO of tricellulin or occludin had limited effects on the morphology and permeability of TJs. Mathematical simulation using a simplified TJ strand network model predicted that reduced cross-links in TJ strands lead to increased permeability of ions and small macromolecules. Furthermore, overexpression of occludin increased the complexity of TJ strand network and strengthened barrier function. Taken together, our data suggest that tricellulin and occludin mediate the formation and/or stabilization of TJ-strand branching points and contribute to the maintenance of epithelial barrier integrity.

Crohn\'s disease (CD) has an altered intestinal barrier function, yet the underlying mechanisms remain to be disclosed. The tricellular tight junction protein tricellulin is involved in the maintenance of the paracellular macromolecule barrier and features an unchanged expression level in CD but a shifted localization. As angulins are known to regulate the localization of tricellulin, we hypothesized the involvement of angulins in CD. Using human biopsies, we found angulin-1 was downregulated in active CD compared with both controls and CD in remission. In T84 and Caco-2 monolayers, leptin, a cytokine secreted by fat tissue and affected in CD, decreased angulin-1 expression. This effect was completely blocked by STAT3 inhibitors, Stattic and WP1066, but only partially by JAK2 inhibitor AG490. The effect of leptin was also seen at a functional level as we observed in Caco-2 cells an increased permeability for FITC-dextran 4 kDa indicating an impaired barrier against macromolecule uptake. In conclusion, we were able to show that in active CD angulin-1 expression is downregulated, which leads to increased macromolecule permeability and is inducible by leptin via STAT3. This suggests that angulin-1 and leptin secretion are potential targets for intervention in CD to restore the impaired intestinal barrier.