Medicinal plant rosemary relaxes blood vessels by activating vascular smooth muscle KCNQ channels.

The evergreen plant rosemary (Salvia rosmarinus) has been employed medicinally for centuries as a memory aid, analgesic, spasmolytic, vasorelaxant and antihypertensive, with recent preclinical and clinical evidence rationalizing some applications. Voltage-gated potassium (Kv) channels in the KCNQ (Kv7) subfamily are highly influential in the nervous system, muscle and epithelia. KCNQ4 and KCNQ5 regulate vascular smooth muscle excitability and contractility and are implicated as antihypertensive drug targets. Here, we found that rosemary extract potentiates homomeric and heteromeric KCNQ4 and KCNQ5 activity, resulting in membrane hyperpolarization. Two rosemary diterpenes, carnosol and carnosic acid, underlie the effects and, like rosemary, are efficacious KCNQ-dependent vasorelaxants, quantified by myography in rat mesenteric arteries. Sex- and estrous cycle stage-dependence of the vasorelaxation matches sex- and estrous cycle stage-dependent KCNQ expression. The results uncover a molecular mechanism underlying rosemary vasorelaxant effects and identify new chemical spaces for KCNQ-dependent vasorelaxants.

LncRNA-BC069792 suppresses tumor progression by targeting KCNQ4 in breast cancer.

BACKGROUND: Breast cancer is the most common malignant tumor that threatens women's health. Attention has been paid on the study of long- non-coding RNA (lncRNA) in breast cancer. However, the specific mechanism remains not clear. METHODS: In this study, we explored the role of lncRNA BC069792 in breast cancer. In vitro and in vivo functional experiments were carried out in cell culture and mouse models. High-throughput next-generation sequencing technology and real-time fluorescence quantitative PCR technology were used to evaluate differentially expressed genes and mRNA expression, Western blot and immunohistochemical staining were used to detect protein expression. RNA immunoprecipitation assay and dual-luciferase activity assay were used to evaluate the competing endogenous RNAs (ceRNA), and rescue and mutation experiments were used for verification. RESULTS: We found that lncRNA BC069792 was expressed at a low level in breast cancer tissues, and significantly decreased in breast cancer with high pathological grade, lymph node metastasis and high Ki-67 index groups. Moreover, BC069792 inhibited the proliferation, invasion and metastasis of breast cancer cells in vitro and in vivo. Mechanically, BC069792 acts as a molecular sponge to adsorb hsa-miR-658 and hsa-miR-4739, to up-regulate the protein expression of Potassium Voltage-Gated Channel Q4 (KCNQ4), inhibits the activities of JAK2 and p-AKT, and plays a role in inhibiting breast cancer growth. CONCLUSIONS: LncRNA BC069792 plays the role of tumor suppressor gene in breast cancer and is a new diagnostic index and therapeutic target in breast cancer.

Valproic Acid Inhibits Progressive Hereditary Hearing Loss in a KCNQ4 Variant Model through HDAC1 Suppression.

Genetic or congenital hearing loss still has no definitive cure. Among genes related to genetic hearing loss, the potassium voltage-gated channel subfamily Q member 4 (KCNQ4) is known to play an essential role in maintaining ion homeostasis and regulating hair cell membrane potential. Variants of the KCNQ4 show reductions in the potassium channel activity and were responsible for non-syndromic progressive hearing loss. KCNQ4 has been known to possess a diverse variant. Among those variants, the KCNQ4 p.W276S variant produced greater hair cell loss related to an absence of potassium recycling. Valproic acid (VPA) is an important and commonly used histone deacetylase (HDAC) inhibitor for class I (HDAC1, 2, 3, and 8) and class IIa (HDAC4, 5, 7, and 9). In the current study, systemic injections of VPA attenuated hearing loss and protected the cochlear hair cells from cell death in the KCNQ4 p.W276S mouse model. VPA activated its known downstream target, the survival motor neuron gene, and increased acetylation of histone H4 in the cochlea, demonstrating that VPA treatment directly affects the cochlea. In addition, treatment with VPA increased the KCNQ4 binding with HSP90ß by inhibiting HDAC1 activation in HEI-OC1 in an in vitro study. VPA is a candidate drug for inhibiting late-onset progressive hereditary hearing loss from the KCNQ4 p.W276S variant.

A humanized murine model, demonstrating dominant progressive hearing loss caused by a novel KCNQ4 mutation (p.G228D) from a large Chinese family.

The pathogenic variants in KCNQ4 cause DFNA2 nonsyndromic hearing loss. However, the understanding of genotype-phenotype correlations between KCNQ4 and hearing is limited. Here, we identified a novel KCNQ4 mutation p.G228D from a Chinese family, including heterozygotes characterized by high-frequency hearing loss that is progressive across all frequencies and homozygotes with more severe hearing loss. We constructed a novel murine model with humanized homologous Kcnq4 mutation. The heterozygotes had mid-frequency and high-frequency hearing loss at 4 weeks, and moved toward all frequencies hearing loss at 12 weeks, while the homozygotes had severe-to-profound hearing loss at 8 weeks. The degeneration of outer hair cells (OHCs) was observed from basal to apical turn of cochlea. The reduced K+ currents and depolarized resting potentials were revealed in OHCs. Remarkably, we observed the loss of inner hair cells (IHCs) in the region corresponding to the frequency above 32 kHz at 8-12 weeks. The results suggest the degeneration of OHCs and IHCs may contribute to high-frequency hearing loss in DFNA2 over time. Our findings broaden the variants of KCNQ4 and provide a novel mouse model of progressive hearing loss, which contributes to an understanding of pathogenic mechanism and eventually treatment of DFNA2 progressive hearing loss.

Activation of KCNQ4 as a Therapeutic Strategy to Treat Hearing Loss.

Potassium voltage-gated channel subfamily q member 4 (KCNQ4) is a voltage-gated potassium channel that plays essential roles in maintaining ion homeostasis and regulating hair cell membrane potential. Reduction of the activity of the KCNQ4 channel owing to genetic mutations is responsible for nonsyndromic hearing loss, a typically late-onset, initially high-frequency loss progressing over time. In addition, variants of KCNQ4 have also been associated with noise-induced hearing loss and age-related hearing loss. Therefore, the discovery of small compounds activating or potentiating KCNQ4 is an important strategy for the curative treatment of hearing loss. In this review, we updated the current concept of the physiological role of KCNQ4 in the inner ear and the pathologic mechanism underlying the role of KCNQ4 variants with regard to hearing loss. Finally, we focused on currently developed KCNQ4 activators and their pros and cons, paving the way for the future development of specific KCNQ4 activators as a remedy for hearing loss.

The agonistic action of URO-K10 on Kv7.4 and 7.5 channels is attenuated by co-expression of KCNE4 ancillary subunit.

KCNQ family constitutes slowly-activating potassium channels among voltage-gated potassium channel superfamily. Recent studies suggested that KCNQ4 and 5 channels are abundantly expressed in smooth muscle cells, especially in lower urinary tract including corpus cavernosum and that both channels can exert membrane stabilizing effect in the tissues. In this article, we examined the electrophysiological characteristics of overexpressed KCNQ4, 5 channels in HEK293 cells with recently developed KCNQ-specific agonist. With submicromolar EC50, the drug not only increased the open probability of KCNQ4 channel but also increased slope conductance of the channel. The overall effect of the drug in whole-cell configuration was to increase maximal whole-cell conductance, to prolongate the activation process, and left-shift of the activation curve. The agonistic action of the drug, however, was highly attenuated by the co-expression of one of the ß ancillary subunits of KCNQ family, KCNE4. Strong in vitro interactions between KCNQ4, 5 and KCNE4 were found through Foster Resonance Energy Transfer and co-immunoprecipitation. Although the expression levels of both KCNQ4 and KCNE4 are high in mesenteric arterial smooth muscle cells, we found that 1 µM of the agonist was sufficient to almost completely relax phenylephrine-induced contraction of the muscle strip. Significant expression of KCNQ4 and KCNE4 in corpus cavernosum together with high tonic contractility of the tissue grants highly promising relaxational effect of the KCNQspecific agonist in the tissue.

[Prediction of KCNQ4gene polymorphism varies with CNE or noise exposure duration on the Risk of NIHL-Cox model analysis based on cohort study].

Objective: The purpose of this study was to explore the association between gene in the potassium recycling pathway 4 (KCNQ4) polymorphisms and the susceptibility to noise-induced hearing loss (NIHL) , and analysis the effect of cumulative noise exposure (CNE) and noise exposure duration on this association. Methods: A nested case-control study with 1∶1 matched was used based on the cohort of noise exposure in a steel factory. A total of 286 cases were selected as the group of hearing loss and 286 controls were chosen according to the matching standards of same gender, same type of work, age difference ≤ 5 years, noise exposure duration ≤ 2 years. The single nucleotide polymorphisms (SNPs) of rs4660468, rs4660470, rs34287852 in KCNQ4 were genotyped by SNPscan(TM) method. The codominant, dominant and recessive models were established to study KCNQ4 polymorphisms and the susceptibility to NIHL by single-factor conditional logistic regression analysis. The COX regression analysis was used to analyze the risk of developing NIHL in individuals with different genotypes along with the extending of noise exposure duration or CNE. Results: In the case of CNE≤96 dB (A) ·year, the risk of developing NIHL in individuals with TA genotype of rs4660470 was 2.197 times than individuals with TT genotypes (95%CI: 1.032~4.677) , and those with TA+AA and TT genotypes (HR=2.467, 95%CI: 1.025~5.934) With the increase of noise exposure duration, in rs4660470, individuals with TA genotype had a higher risk of suffering NIHL than those with TT genotype (HR=1.461, 95%CI: 1.061~2.011) , individuals with TA and/or AA genotype had a earlier risk of suffering NIHL than those with TT genotype. Conclusion: The mutant allele A of rs4660470 in KCNQ4 may be a risk factor for developing NIHL, CNE≤100 dB (A) ·year or the increase of noise exposure duration may further increase the risk of NIHL.

A novel pore-region mutation, c.887G > A (p.G296D) in KCNQ4, causing hearing loss in a Chinese family with autosomal dominant non-syndromic deafness 2.

BACKGROUND: Hereditary non-syndromic hearing loss is the most common inherited sensory defect in humans. The KCNQ4 channel belongs to a family of potassium ion channels that play crucial roles in physiology and disease. Mutations in KCNQ4 underlie deafness non-syndromic autosomal dominant 2, a subtype of autosomal dominant, progressive, high-frequency hearing loss. METHODS: A six-generation Chinese family from Hebei Province with autosomal dominantly inherited, sensorineural, postlingual, progressive hearing loss was enrolled in this study. Mutation screening of 129 genes associated with hearing loss was performed in five family members by next-generation sequencing (NGS). We also carried out variant analysis on DNA from 531 Chinese individuals with normal hearing as controls. RESULTS: This family exhibits postlingual, progressive, symmetrical, bilateral, non-syndromic sensorineural hearing loss. NGS, bioinformatic analysis, and Sanger sequencing confirmed the co-segregation of a novel mutation [c.887G > A (p.G296D)] in KCNQ4 with the disease phenotype in this family. This mutation leads to a glycine-to-aspartic acid substitution at position 296 in the pore region of the KCNQ4 channel. This mutation affects a highly conserved glutamic acid. NGS is a highly efficient tool for identifying gene mutations causing heritable disease. CONCLUSIONS: Progressive hearing loss is common in individuals with KCNQ4 mutations. NGS together with Sanger sequencing confirmed that the five affected members of this Chinese family inherited a missense mutation, c.887G > A (p.G296D), in exon 6 of KCNQ4. Our results increase the number of identified KCNQ4 mutations.

Novel exon 1 protein-coding regions N-terminally extend human KCNE3 and KCNE4.

The 5 human (h)KCNE ß subunits each regulate various cation channels and are linked to inherited cardiac arrhythmias. Reported here are previously undiscovered protein-coding regions in exon 1 of hKCNE3 and hKCNE4 that extend their encoded extracellular domains by 44 and 51 residues, which yields full-length proteins of 147 and 221 residues, respectively. Full-length hKCNE3 and hKCNE4 transcript and protein are expressed in multiple human tissues; for hKCNE4, only the longer protein isoform is detectable. Two-electrode voltage-clamp electrophysiology revealed that, when coexpressed in Xenopus laevis oocytes with various potassium channels, the newly discovered segment preserved conversion of KCNQ1 by hKCNE3 to a constitutively open channel, but prevented its inhibition of Kv4.2 and KCNQ4. hKCNE4 slowing of Kv4.2 inactivation and positive-shifted steady-state inactivation were also preserved in the longer form. In contrast, full-length hKCNE4 inhibition of KCNQ1 was limited to 40% at +40 mV vs. 80% inhibition by the shorter form, and augmentation of KCNQ4 activity by hKCNE4 was entirely abolished by the additional segment. Among the genome databases analyzed, the longer KCNE3 is confined to primates; full-length KCNE4 is widespread in vertebrates but is notably absent from Mus musculus Findings highlight unexpected KCNE gene diversity, raise the possibility of dynamic regulation of KCNE partner modulation via splice variation, and suggest that the longer hKCNE3 and hKCNE4 proteins should be adopted in future mechanistic and genetic screening studies.-Abbott, G. W. Novel exon 1 protein-coding regions N-terminally extend human KCNE3 and KCNE4.

A novel frameshift mutation in KCNQ4 in a family with autosomal recessive non-syndromic hearing loss.

Mutation of KCNQ4 has been reported to cause autosomal dominant non-syndromic hearing loss (DFNA2A) that usually presents as progressive hearing loss starting from mild to moderate hearing loss during childhood. Here, we identified a novel KCNQ4 mutation, c.1044_1051del8, in a family with autosomal recessive non-syndromic hearing loss. The proband was homozygous for the mutation and was born to consanguineous parents; she showed severe hearing loss that was either congenital or of early childhood onset. The proband had a sister who was heterozygous for the mutation but showed normal hearing. The mutation caused a frameshift that eliminated most of the cytoplasmic C-terminus, including the A-domain, which has an important role for protein tetramerization, and the B-segment, which is a binding site for calmodulin (CaM) that regulates channel function via Ca ions. The fact that the heterozygote had normal hearing indicates that sufficient tetramerization and CaM binding sites were present to preserve a normal phenotype even when only half the proteins contained an A-domain and B-segment. On the other hand, the severe hearing loss in the homozygote suggests that complete loss of the A-domain and B-segment in the protein caused loss of function due to the failure of tetramer formation and CaM binding. This family suggests that some KCNQ4 mutations can cause autosomal recessive hearing loss with more severe phenotype in addition to autosomal dominant hearing loss with milder phenotype. This genotype-phenotype correlation is analogous to that in KCNQ1 which causes autosomal dominant hereditary long QT syndrome 1 with milder phenotype and the autosomal recessive Jervell and Lange-Nielsen syndrome 1 with more severe phenotype due to deletion of the cytoplasmic C-terminus of the potassium channel.

Cloning, characterization, and evolutionary patterns of KCNQ4 genes in anurans.

Acoustic communication plays important roles in the survival and reproduction of anurans. The perception and discrimination of conspecific sound signals of anurans were always affected by masking background noise. Previous studies suggested that some frogs evolved the high-frequency hearing to minimize the low-frequency noise. However, the molecular mechanisms underlying the high-frequency hearing in anurans have not been well explored. Here, we cloned and obtained the coding regions of a high-frequency hearing-related gene (KCNQ4) from 11 representative anuran species and compared them with orthologous sequences from other four anurans. The sequence characteristics and evolutionary analyses suggested the highly conservation of the KCNQ4 gene in anurans, which supported their functional importance. Branch-specific analysis showed that KCNQ4 genes were under different evolutionary forces in anurans and most anuran lineages showed a generally strong purifying selection. Intriguingly, one significantly positively selected site was identified in the anuran KCNQ4 gene based on FEL model. Positive selection was also found along the common ancestor of Ranidae and Rhacophoridae as well as the ancestral O. tianmuii based on the branch-site analysis, and the positively selected sites identified were involved in or near the N-terminal ion transport and the potassium ion channel functional domain of the KCNQ4 genes. The present study revealed valuable information regarding the KCNQ4 genes in anurans and provided some new insights for the underpinnings of the high-frequency hearing in frogs.

Natural History of KCNQ4 p.G285S Related Hearing Loss, Construction of iPSC and Mouse Model.

OBJECTIVE: KCNQ4 is one of the most common disease-causing genes involved in autosomal dominant non-syndromic hearing loss. We previously found that patients with KCNQ4 p.G285S exhibited a much more rapid deterioration in hearing loss than those with other KCNQ4 variants. To determine the rate of hearing loss and assess the disease for further analysis, we performed a long-term follow-up of these patients and generated patient-derived induced pluripotent stem cells (iPSCs), and a mouse model. METHODS: Patients with KCNQ4 p.G285S from a five-generation family with hearing loss were followed up from 2005 to 2022. iPSCs were generated by stimulating peripheral blood mononuclear cells from the proband, and their pluripotency was determined. The Kcnq4 p.G286S mouse model was generated using CRISPR/Cas9, and its genotype and phenotype were identified. RESULTS: (1) The annual rates of hearing loss at the frequencies of speech were 0.96 dB for the proband and 0.87 dB for his father during the follow-up period, which were faster than patients with other KCNQ4 variants. (2) The patient-derived iPSC line carrying KCNQ4 p.G285S, possessed the capacity of differentiation and pluripotency capacities. (3) Mutant mice with Kcnq4 p.G286S exhibited hearing loss and outer hair cell loss at 1 month of age. CONCLUSION: Patients with KCNQ4 p.G285S variant exhibited significantly accelerated progression of hearing loss compared to those with other reported variants. Awareness of the natural history of hearing loss associated with KCNQ4 p.G285S is beneficial for genetic counseling and prognosis. The generation of the iPSCs and mouse model can provide a valuable foundation for further in-depth analyses. LEVEL OF EVIDENCE: 4 Laryngoscope, 134:2356-2363, 2024.

A novel variant c.902C>A (p. A301D) in KCNQ4 associated with non-syndromic deafness 2A in a Chinese family.

BACKGROUND: Deafness autosomal dominant 2A (DFNA2A) is related to non-syndromic genetic hearing impairment. The KCNQ4 (Potassium Voltage-Gated Channel Subfamily Q Member 4) can lead to DFNA2A. In this study, we report a case of autosomal dominant non-syndromic hearing loss with six family members as caused by a novel variant in the KCNQ4 gene. METHODS: The whole-exome sequencing (WES) and pure tone audiometry were performed on the proband of the family. Sanger sequencing was conducted on family members to determine if the novel variant in the KCNQ4 gene was present. Evolutionary conservation analysis and computational tertiary structure protein prediction of the wild-type KCNQ4 protein and its variant were then performed. In addition, voltage-gated channel activity of the wild-type KCNQ4 protein and its variant were tested using whole-cell patch clamp. RESULTS: It was observed that the proband had inherited autosomal dominant, non-syndromic sensorineural hearing loss as a trait. A novel co-segregating heterozygous missense variant (c.902C>A, p.Ala301Asp) of the KCNQ4 gene was identified in the proband and other five affected family members. This variant was predicted to cause an alanine-to-aspartic acid substitution at position 301 in the KCNQ4 protein. The alanine at position 301 is well conserved across different species. Whole-cell patch clamp showed that there was a significant difference between the WT protein currents and the mutant protein currents in the voltage-gated channel activity. CONCLUSION: In the present study, performing WES in conjunction with Sanger sequencing enhanced the detection of a novel, potentially causative variant (c301 A>G; p.Ala301Asp) in exon 6 of the KCNQ4 gene. Therefore, our findings contributed to the mutation spectrum of the KCNQ4 gene and may be useful in the diagnosis and gene therapy of deafness autosomal dominant 2A.

Impaired surface expression and conductance of the KCNQ4 channel lead to sensorineural hearing loss.

KCNQ4, a voltage-gated potassium channel, plays an important role in maintaining cochlear ion homoeostasis and regulating hair cell membrane potential, both essential for normal auditory function. Mutations in the KCNQ4 gene lead to DFNA2, a subtype of autosomal dominant non-syndromic deafness that is characterized by progressive sensorineural hearing loss across all frequencies. Despite recent advances in the identification of pathogenic KCNQ4 mutations, the molecular aetiology of DFNA2 remains unknown. We report here that decreased cell surface expression and impaired conductance of the KCNQ4 channel are two mechanisms underlying hearing loss in DFNA2. In HEK293T cells, a dramatic decrease in cell surface expression was detected by immunofluorescent microscopy and confirmed by Western blot for the pathogenic KCNQ4 mutants L274H, W276S, L281S, G285C, G285S, G296S and G321S, while their overall cellular levels remained normal. In addition, none of these mutations affected tetrameric assembly of KCNQ4 channels. Consistent with these results, all mutants showed strong dominant-negative effects on the wild-type (WT) channel function. Most importantly, overexpression of HSP90ß, a key component of the molecular chaperone network that controls the KCNQ4 biogenesis, significantly increased cell surface expression of the KCNQ4 mutants L281S, G296S and G321S. KCNQ4 surface expression was restored or considerably improved in HEK293T cells mimicking the heterozygous condition of these mutations in DFNA2 patients. Finally, our electrophysiological studies demonstrated that these mutations directly compromise the conductance of the KCNQ4 channel, since no significant change in KCNQ4 current was observed after KCNQ4 surface expression was restored or improved.

[The genotype-phenotype correlation analysis and genetic counseling of hearing loss patients with novel KCNQ4 mutations].

Objective:To provide accurate genetic counseling, the genotype-phenotype correlation of the patients with KCNQ4mutations was analyzed. Methods:Two hearing loss families, 1807956（a five-generation family with 34 members） and 1707806（a three-generation family with 12 members） were recruited. The candidate variants were detected by next generation sequencing technology. Sanger sequencing was performed to verify the co-segregation of the phenotype in the recruited family members. According to American College of Medical Genetics and Genomics（ACMG） guideline, combined with clinical data, genetic testing, bioinformatic analysis and electrophysiological experiments, the pathogenicity of mutations was analyzed and genetic counseling was provided for family members. Results:The proband of family 1807956 was a pregnant woman, who carried KCNQ4 c.808T>G p.Y270D and developed hearing loss at the age of 15 years old, she had profound hearing loss in both ears, with middle-frequency highly affected. The proband of family 1707806 was an adolescent whose onset age was 11 years old, carrying KCNQ4 c.733G>A p.G245R, he presented with bilateral moderately severe hearing loss. The inheritance pattern of these two families were autosomal dominant inheritance. The two variants were missense mutations that were co-segregation in the two families and were not found in normal population. The mutations predicted by bioinformatic analysis tools were damaging and highly conserved in different species. Electrophysiological experiments showed that the function of the mutant ion channels was impaired. According to ACMG guideline, KCNQ4 c.808T>G was pathogenic, and KCNQ4 c.733G>A was likely pathogenic. Conclusion:The two mutations in this research were reported for the first time. The hearing loss of the patients showed heterogeneity, enriching the variation spectrum and clinical phenotype of KCNQ4.

In vivo outer hair cell gene editing ameliorates progressive hearing loss in dominant-negative Kcnq4 murine model.

Outer hair cell (OHC) degeneration is a major cause of progressive hearing loss and presbycusis. Despite the high prevalence of these disorders, targeted therapy is currently not available. Methods: We generated a mouse model harboring Kcnq4W276S/+ to recapitulate DFNA2, a common genetic form of progressive hearing loss accompanied by OHC degeneration. After comprehensive optimization of guide RNAs, Cas9s, vehicles, and delivery routes, we applied in vivo gene editing strategy to disrupt the dominant-negative allele in Kcnq4 and prevent progressive hearing loss. Results:In vivo gene editing using a dual adeno-associated virus package targeting OHCs significantly improved auditory thresholds in auditory brainstem response and distortion-product otoacoustic emission. In addition, we developed a new live-cell imaging technique using thallium ions to investigate the membrane potential of OHCs and successfully demonstrated that mutant allele disruption resulted in more hyperpolarized OHCs, indicating elevated KCNQ4 channel activity. Conclusion: These findings can facilitate the development of targeted therapies for DFNA2 and support the use of CRISPR-based gene therapy to rectify defects in OHCs.

Precise detection of CRISPR-Cas9 editing in hair cells in the treatment of autosomal dominant hearing loss.

Gene therapy would benefit from the effective editing of targeted cells with CRISPR-Cas9 tools. However, it is difficult to precisely assess the editing performance in vivo because the tissues contain many non-targeted cells, which is one of the major barriers to clinical translation. Here, in the Atoh1-GFP;Kcnq4 +/G229D mice, recapitulating a novel mutation we identified in a hereditary hearing loss pedigree, the high-efficiency editing of CRISPR-Cas9 in hair cells (34.10% on average) was precisely detected by sorting out labeled cells compared with only 1.45% efficiency in the whole cochlear tissue. After injection of the developed AAV_SaCas9-KKH_sgRNA agents, the Kcnq4 +/G229D mice showed significantly lower auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) thresholds, shorter ABR wave I latencies, higher ABR wave I amplitudes, increased number of surviving outer hair cells (OHCs), and more hyperpolarized resting membrane potentials of OHCs. These findings provide an innovative approach to accurately assess the underestimated editing efficiency of CRISPR-Cas9 in vivo and offer a promising strategy for the treatment of KCNQ4-related deafness.

The Pathological Mechanisms of Hearing Loss Caused by KCNQ1 and KCNQ4 Variants.

Deafness-associated genes KCNQ1 (also associated with heart diseases) and KCNQ4 (only associated with hearing loss) encode the homotetrameric voltage-gated potassium ion channels Kv7.1 and Kv7.4, respectively. To date, over 700 KCNQ1 and over 70 KCNQ4 variants have been identified in patients. The vast majority of these variants are inherited dominantly, and their pathogenicity is often explained by dominant-negative inhibition or haploinsufficiency. Our recent study unexpectedly identified cell-death-inducing cytotoxicity in several Kv7.1 and Kv7.4 variants. Elucidation of this cytotoxicity mechanism and identification of its modifiers (drugs) have great potential for aiding the development of a novel pharmacological strategy against many pathogenic KCNQ variants. The purpose of this review is to disseminate this emerging pathological role of Kv7 variants and to underscore the importance of experimentally characterizing disease-associated variants.

miR-153/KCNQ4 axis contributes to noise-induced hearing loss in a mouse model.

Damage to the cochlear sensory epithelium is a key contributor to noise-induced sensorineural hearing loss (SNHL). KCNQ4 plays an important role in the cochlear potassium circulation and outer hair cells survival. As miR-153 can target and regulate KCNQ4, we sought to study the role of miR-153 in SNHL. 12-week-old male CBA/J mice were exposed to 2-20 kHz broadband noise at 96 dB SPL to induce temporary threshold shifts and 101 dB SPL to induce permanent threshold shifts. Hearing loss was determined by auditory brainstem responses (ABR). Relative expression of miR-153 and KCNQ4 in mice cochlea were determined by Real-Time quantitative PCR. miR-153 mimics were co-transfected with wild type or mutated KCNQ4 into HEK293 cells. Luciferase reporter assay was used to validate the binding between miR-153 and KCNQ4. AAV-sp-153 was constructed and administrated intra-peritoneally 24- and 2-h prior and immediately after noise exposure to knockdown miR-153. The KCNQ4 is mainly expressed in outer hair cells (OHCs). We showed that the expression of KCNQ4 in mice cochlea was reduced and miR-153 expression was significantly increased after noise exposure compared to control. miR-153 bound to 3'UTR of KNCQ4, and the knockdown of miR-153 with the AAV-sp-153 administration restored KCNQ4 mRNA and protein expression. In addition, the knockdown of miR-153 reduced ABR threshold shifts at 8, 16, and 32 kHz after permanent threshold shifts (PTS) noise exposure. Correspondingly, OHC losses were attenuated with inhibition of miR-153. This study demonstrates that miR-153 inhibition significantly restores KNCQ4 in cochlea after noise exposure, which attenuates SNHL. Our study provides a new potential therapeutic target in the prevention and treatment of SNHL.

A KCNQ4 c.546C>G Genetic Variant Associated with Late Onset Non-Syndromic Hearing Loss in a Taiwanese Population.

Clinical presentation is heterogeneous for autosomal dominant nonsyndromic hearing loss (ADNSHL). Variants of KCNQ4 gene is a common genetic factor of ADNSHL. Few studies have investigated the association between hearing impairment and the variant c.546C>G of KCNQ4. Here, we investigated the phenotype and clinical manifestations of the KCNQ4 variant. Study subjects were selected from the participants of the Taiwan Precision Medicine Initiative. In total, we enrolled 12 individuals with KCNQ4 c.546C>G carriers and 107 non-carriers, and performed pure tone audiometry (PTA) test and phenome-wide association (PheWAS) analysis for the patients. We found that c.546C>G variant was related to an increased risk of hearing loss. All patients with c.546C>G variant were aged >65 years and had sensorineural and high frequency hearing loss. Of these patients, a third (66.7%) showed moderate and progressive hearing loss, 41.7% complained of tinnitus and 16.7% complained of vertigo. Additionally, we found a significant association between KCNQ4 c.546C>G variant, aortic aneurysm, fracture of lower limb and polyneuropathy in diabetes. KCNQ4 c.546C>G is likely a potentially pathogenic variant of ADNSHL in the elderly population. Genetic counseling, annual audiogram and early assistive listening device intervention are highly recommended to prevent profound hearing impairment in this patient group.