Novel KCNQ4 variants in different functional domains confer genotype- and mechanism-based therapeutics in patients with nonsyndromic hearing loss.

Loss-of-function variant in the gene encoding the KCNQ4 potassium channel causes autosomal dominant nonsyndromic hearing loss (DFNA2), and no effective pharmacotherapeutics have been developed to reverse channel activity impairment. Phosphatidylinositol 4,5-bisphosphate (PIP2), an obligatory phospholipid for maintaining KCNQ channel activity, confers differential pharmacological sensitivity of channels to KCNQ openers. Through whole-exome sequencing of DFNA2 families, we identified three novel KCNQ4 variants related to diverse auditory phenotypes in the proximal C-terminus (p.Arg331Gln), the C-terminus of the S6 segment (p.Gly319Asp), and the pore region (p.Ala271_Asp272del). Potassium currents in HEK293T cells expressing each KCNQ4 variant were recorded by patch-clamp, and functional recovery by PIP2 expression or KCNQ openers was examined. In the homomeric expression setting, the three novel KCNQ4 mutant proteins lost conductance and were unresponsive to KCNQ openers or PIP2 expression. Loss of p.Arg331Gln conductance was slightly restored by a tandem concatemer channel (WT-p.R331Q), and increased PIP2 expression further increased the concatemer current to the level of the WT channel. Strikingly, an impaired homomeric p.Gly319Asp channel exhibited hyperactivity when a concatemer (WT-p.G319D), with a negative shift in the voltage dependence of activation. Correspondingly, a KCNQ inhibitor and chelation of PIP2 effectively downregulated the hyperactive WT-p.G319D concatemer channel. Conversely, the pore-region variant (p.Ala271_Asp272del) was nonrescuable under any condition. Collectively, these novel KCNQ4 variants may constitute therapeutic targets that can be manipulated by the PIP2 level and KCNQ-regulating drugs under the physiological context of heterozygous expression. Our research contributes to the establishment of a genotype/mechanism-based therapeutic portfolio for DFNA2.

Whole-exome sequencing identifies two novel mutations in KCNQ4 in individuals with nonsyndromic hearing loss.

Mutations in potassium voltage-gated channel subfamily Q member 4 (KCNQ4) are etiologically linked to a type of nonsyndromic hearing loss, deafness nonsyndromic autosomal dominant 2 (DFNA2). We performed whole-exome sequencing for 98 families with hearing loss and found mutations in KCNQ4 in five families. In this study, we characterized two novel mutations in KCNQ4: a missense mutation (c.796G>T; p.Asp266Tyr) and an in-frame deletion mutation (c.259_267del; p.Val87_Asn89del). p.Asp266Tyr located in the channel pore region resulted in early onset and moderate hearing loss, whereas p.Val87_Asn89del located in the N-terminal cytoplasmic region resulted in late onset and high frequency-specific hearing loss. When heterologously expressed in HEK 293 T cells, both mutant proteins did not show defects in protein trafficking to the plasma membrane or in interactions with wild-type (WT) KCNQ4 channels. Patch-clamp analysis demonstrated that both p.Asp266Tyr and p.Val87_Asn89del mutant channels lost conductance and were completely unresponsive to KCNQ activators, such as retigabine, zinc pyrithione, and ML213. Channels assembled from WT-p.Asp266Tyr concatemers, like those from WT-WT concatemers, exhibited conductance and responsiveness to KCNQ activators. However, channels assembled from WT-p.Val87_Asn89del concatemers showed impaired conductance, suggesting that p.Val87_Asn89del caused complete loss-of-function with a strong dominant-negative effect on functional WT channels. Therefore, the main pathological mechanism may be related to loss of K+ channel activity, not defects in trafficking.