*5.3.1 Solute carrier family 12 member 2 (SLC12A2)*

SLC12A2 also referred to as Na<sup>+</sup> K+ 2xCl− co-transporter (NKCC1) is a member of solute carrier family 12 which is involved in the transport of sodium, potassium, and chloride ions across the secretory and absorptive epithelia [124]. It is found to be expressed in the basolateral membrane of marginal strial cells and in the lateral wall fibrocytes in cochlea of rodents and non-human primates. Fibrocytes take up K<sup>+</sup> from perilymph through SLC12A2 and ATP1A2 (α2-Na<sup>+</sup> K+ ATPase) [125, 126] and pass it to intermediate cells and strial basal cells through gap junctions [127]. It also transports K+ from the intra-strial space into the marginal cells [128]. In mice, it has been shown that heterozygous variants in *Slc12a2* can result in progressive ARHL without causing

any damage to the cochlear morphology [129]. However, the role of this gene for ARHL in humans has not been tested yet.

#### *5.3.2 Voltage gated potassium channel KQT like subfamily member 4 (KCNQ4)*

Potassium channels play an important role in maintaining ionic composition and electrical signaling in biological fluids. Cochlear hair cells and vestibular structures have voltage-gated potassium channels encoded by *KCNQ4* to maintain ionic balance in cochlear fluid [130]. Missense variants in *KCNQ4* are commonly known to cause non-syndromic autosomal dominant hearing loss, DFNA2 [131–134]. However, in a study of two Caucasian populations, several SNPs were found associated with ARHL, all of which were localized to a 13 kb region in the middle of the *KCNQ4* [135].

#### *5.3.3 Wolframin (WFS1)*

*WFS1* encodes Wolframin which is a transmembrane protein and is thought to be a large cation-selective ion channel [136]. Variants in *WFS1* commonly cause Wolfram syndrome [137], and non-syndromic autosomal dominant low frequency sensorineural hearing loss [138–140]. In 2017, a study comprising 518 Finnish adults showed a heterozygous variant p.(Tyr528His) associated with late-onset hearing loss. Most of the individuals participating in this cohort initially had hearing loss which affected the high frequencies and subsequently progressed to involve middle and low frequencies [141].

#### *5.3.4 Solute carrier family 7 member 8 (SLC7A8)*

SLC7A8 functions as a sodium-independent transporter of L-type amino acids in many organs in vertebrates [142]. It is highly expressed in the inner ear [143, 144], and specifically localizes to the stria vascularis [143]. A study focused on the role of *Slc7a8* in ARHL using mouse models demonstrated that in homozygous knockout mice (*Slc7a8*−/−*)* the loss of function leads to high-frequency hearing loss which progressively extends to low frequencies. While, interestingly, the young knockout mice heterozygous for *Slc7a8* (*Slc7a8*+/−) did not show a hearing loss. However, with aging, these mice developed high-frequency hearing loss earlier than the wild-type mice [144]. Similarly, in a cohort of 66 ARHL patients from Italy, genome sequencing identified four heterozygous variants (p.Val302Ile, p.Arg418His, p.Thr402Met and p.Val460Glu) in SLC7A8 [144]. In vitro functional studies of these variants further confirmed a significant decrease in amino acid transport activity supporting *SLC7A8* as a causative gene for ARHL.

#### **5.4 Mitochondrial antioxidative enzymes**

Mitochondrial DNA (mtDNA) variants have long been known to cause various human diseases, including non-syndromic hearing loss [145]. A significant increase in the contribution of mtDNA variants has been observed in aging human auditory system [146]. The postmortem analyses of human temporal bones have shown a 4977 bp deletion (also known as common deletion (CD)) in mtDNA as a frequent cause of ARHL [147, 148]. Additionally, a decrease in the expression of another mitochondrial enzyme cytochrome oxidase 3 (*COX3*) in spiral ganglion cells was also reported in ARHL patients [149].

Isocitrate dehydrogenase (*IDH*) is a key component of the aerobic metabolism in mitochondria, which facilitates the generation of NADPH and NADH thus regulating the cellular oxidative stress [150, 151]. It has been demonstrated in a mouse study that the expression of *IDH2* normally decreases with age and when this gene was knocked out there was increased oxidative stress in the murine inner ear leading to the loss of hair cells and damage to spiral ganglion [152].
