Overlooked KCNQ4 variants augment the risk of hearing loss.

Pathogenic variants of KCNQ4 cause symmetrical, late-onset, progressive, high-frequency-affected hearing loss, which eventually involves all frequencies with age. To understand the contribution of KCNQ4 variants to hearing loss, we analyzed whole-exome and genome sequencing data from patients with hearing loss and individuals whose hearing phenotypes were unknown. In KCNQ4, we identified seven missense variants and one deletion variant in 9 hearing loss patients and 14 missense variants in the Korean population with an unknown hearing loss phenotype. The p.R420W and p.R447W variants were found in both cohorts. To investigate the effects of these variants on KCNQ4 function, we performed whole-cell patch clamping and examined their expression levels. Except for p.G435Afs*61, all KCNQ4 variants exhibited normal expression patterns similar to those of wild-type KCNQ4. The p.R331Q, p.R331W, p.G435Afs*61, and p.S691G variants, which were identified in patients with hearing loss, showed a potassium (K+) current density lower than or similar to that of p.L47P, a previously reported pathogenic variant. The p.S185W and p.R216H variants shifted the activation voltage to hyperpolarized voltages. The channel activity of the p.S185W, p.R216H, p.V672M, and p.S691G KCNQ4 proteins was rescued by the KCNQ activators retigabine or zinc pyrithione, whereas p.G435Afs*61 KCNQ4 proteins were partially rescued by sodium butyrate, a chemical chaperone. Additionally, the structure of the variants predicted using AlphaFold2 showed impaired pore configurations, as did the patch-clamp data. Our findings suggest that KCNQ4 variants may be overlooked in hearing loss that starts in adulthood. Some of these variants are medically treatable; hence, genetic screening for KCNQ4 is important.

OSBPL2 mutations impair autophagy and lead to hearing loss, potentially remedied by rapamycin.

Intracellular accumulation of mutant proteins causes proteinopathies, which lack targeted therapies. Autosomal dominant hearing loss (DFNA67) is caused by frameshift mutations in OSBPL2. Here, we show that DFNA67 is a toxic proteinopathy. Mutant OSBPL2 accumulated intracellularly and bound to macroautophagy/autophagy proteins. Consequently, its accumulation led to defective endolysosomal homeostasis and impaired autophagy. Transgenic mice expressing mutant OSBPL2 exhibited hearing loss, but osbpl2 knockout mice or transgenic mice expressing wild-type OSBPL2 did not. Rapamycin decreased the accumulation of mutant OSBPL2 and partially rescued hearing loss in mice. Rapamycin also partially improved hearing loss and tinnitus in individuals with DFNA67. Our findings indicate that dysfunctional autophagy is caused by mutant proteins in DFNA67; hence, we recommend rapamycin for DFNA67 treatment.Abbreviations: ABR: auditory brainstem response; ACTB: actin beta; CTSD: cathepsin D; dB: decibel; DFNA67: deafness non-syndromic autosomal dominant 67; DPOAE: distortion product otoacoustic emission; fs: frameshift; GFP: green fluorescent protein; HsQ53R-TG: human p.Q53Rfs*100-transgenic: HEK 293: human embryonic kidney 293; HFD: high-fat diet; KO: knockout; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NSHL: non-syndromic hearing loss; OHC: outer hair cells; OSBPL2: oxysterol binding protein-like 2; SEM: scanning electron microscopy; SGN: spiral ganglion neuron; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TG: transgenic; WES: whole-exome sequencing; YUHL: Yonsei University Hearing Loss; WT: wild-type.

COCH-related autosomal dominant nonsyndromic hearing loss: a phenotype-genotype study.

This phenotype-genotype study aimed to investigate the extent of audioprofile variability related to cochlin major domains and to identify potential ethnic-specific differences associated with COCH-related hearing loss. Eight Korean families (26 cases) were diagnosed with COCH-related hearing loss by exome sequencing. Audiometric test results were combined with those from nine published East Asian families (20 cases) and compared with those from 38 European-descent families (277 cases). Audioprofiles were created by grouping audiometric test results into age ranges by age at testing and then averaging hearing loss thresholds by frequency within age ranges. The functional impact of the identified variants was assessed in vitro by examining the intracellular trafficking, secretion, and cleavage of cochlin. In both East Asian and European-descent families segregating COCH-related hearing loss, deafness-associated variants in non-LCCL domains of cochlin were associated with hearing loss that was more severe earlier in life than hearing loss caused by variants in the LCCL domain. Consistent with this phenotypic difference, functional studies demonstrated distinct pathogenic mechanisms for COCH variants in a domain-dependent manner; specifically, a cytotoxic effect was observed for the p.Phe230Leu variant, which is located in the vWFA1 domain. No ethnic-specific differences in hearing loss progression were observed, except for those attributable to an overrepresentation of presymptomatic cases in the European-descent cohort.

Differential genetic diagnoses of adult post-lingual hearing loss according to the audiogram pattern and novel candidate gene evaluation.

Ski-slope hearing loss (HL), which refers to increased auditory threshold at high frequencies, is common in adults. However, genetic contributions to this post-lingual HL remain largely unknown. Here, we prospectively investigated deafness-associated and novel candidate genes causing ski-slope HL. We analyzed 192 families with post-lingual HL via gene panel and/or exome sequencing. With an overall molecular diagnostic rate of 35.4% (68/192) in post-lingual HL, ski-slope HL showed a lower diagnostic rate (30.7%) compared with other conditions (40.7%). In patients who showed HL onset before the age of 40, genetic diagnostic probability was significantly lower for ski-slope HL than for other conditions. Further analysis of 51 genetically undiagnosed patients in the ski-slope HL group identified three variants in delta-like ligand 1 (DLL1), a Notch ligand, which presented in vitro gain-of-function effects on Notch downstream signaling. In conclusion, genetic diagnostic rates in post-lingual HL varied according to audiogram patterns with age-of-onset as a confounding factor. DLL1 was identified as a candidate gene causing ski-slope HL.

Theoretical studies on the mechanism of acid-promoted hydrolysis of N-formylaziridine in comparison with formamide.

[reaction: see text] We present an ab initio study of the acid-promoted hydrolysis reaction mechanism of N-formylaziridine in comparison with formamide. Since the rate of amide hydrolysis reactions depends on the formation of the tetrahedral intermediate, we focused our attention mainly on the reactant complex, the tetrahedral intermediate, and the transition state connecting these two stationary points. Geometries were optimized using the density functional theory, and the energetics were refined using ab initio theory including electron correlation. Solvent effects were investigated by using polarizable continuum method calculations. The proton-transfer reaction between the O-protonated and N-protonated amides was investigated. In acidic media, despite that the N-protonated species is more stable than the O-protonated one, it is predicted that both N-protonated and O-protonated pathways compete in the hydrolysis reaction of N-formylaziridine.

Molecular recognition of fluoride anion: benzene-based tripodal imidazolium receptor.

A benzene-based tripodal imidazolium receptor utilizing the strong (C-H)(+)...X(-) hydrogen bonding interaction between imidazolium moieties and halide anions is extensively investigated both theoretically and experimentally. Ab initio calculations predict that this receptor has a very high affinity for fluoride ion (F(-)). The association constant and free energy gain of the N-butyl receptor 2 for F(-) in acetonitrile were measured to be 2.1 x 10(5) M(-1) and -7.25 kcal/mol, respectively, showing that the receptor has a high affinity for F(-) in highly polar organic solvents.

Rational design of biologically important chemosensors: a novel receptor for selective recognition of acetylcholine over ammonium cations.

[structure: see text] In consideration of competition between cation-pi and hydrogen bond interaction forces, we designed a novel receptor, 1,3,5-tris(pyrrolyl)benzene, which shows high selectivity for acetylcholine (ACh). The selectivity of the receptor for ACh over other ammonium cations is demonstrated by the ion-selective electrode (ISE) method in buffer solution. The binding free energy of the receptor with ACh in chloroform solution is measured to be 3.65 kcal/mol in the presence of chloride anion by nuclear magnetic resonance spectroscopy, and that in water is estimated to be much greater ( approximately 6 kcal/mol).

Catalytic mechanism of enzymes: preorganization, short strong hydrogen bond, and charge buffering.

During the past decade, there has been much debate about the enormous catalytic rate enhancement observed in enzymatic reactions involving carbanion intermediates. Our recent theoretical study has demonstrated the importance of the short strong hydrogen bond (SSHB) in the enzymatic reactions. Nevertheless, other recent theoretical studies espouse the role of preorganization over that of the SSHB. To achieve a consensus on this issue and to find the truth, a more clarified explanation must be given. To this end, we have carried out an elaborate analysis of these enzymatic reactions. We here clarify that the catalytic mechanism needs to be explained with three important factors, viz., SSHB, preorganization, and charge buffering/dissipation. Since the charge buffering role is different from the commonly used concepts of the SSHB and preorganization (unless these definitions are expanded), we stress that the charge buffering role of the catalytic residues is an important ingredient of the enzymatic reaction in reducing the level of accumulation of the negative charge on the substrate during the reaction process. This charge reduction is critical to the lowering of activation barriers and the stabilization of intermediates.