Gipc3 mutations associated with audiogenic seizures and sensorineural hearing loss in mouse and human

Sensorineural hearing loss affects the quality of life and communication of millions of people, but the underlying molecular mechanisms remain elusive. Here, we identify mutations in Gipc3 underlying progressive sensorineural hearing loss (age-related hearing loss 5, ahl5) and audiogenic seizures (juvenile audiogenic monogenic seizure 1, jams1) in mice and autosomal recessive deafness DFNB15 and DFNB95 in humans. Gipc3 localizes to inner ear sensory hair cells and spiral ganglion. A missense mutation in the PDZ domain has an attenuating effect on mechanotransduction and the acquisition of mature inner hair cell potassium currents. Magnitude and temporal progression of wave I amplitude of afferent neurons correlate with susceptibility and resistance to audiogenic seizures. The Gipc3343A allele disrupts the structure of the stereocilia bundle and affects long-term function of auditory hair cells and spiral ganglion neurons. Our study suggests a pivotal role of Gipc3 in acoustic signal acquisition and propagation in cochlear hair cells.

D egeneration of the sensory hair cells in the organ of Corti and their innervating aff erent neurons in the cochlear spiral ganglion results in sensorineural hearing loss (SNHL), which typically begins at the higher frequencies ( > 15 kHz) and gradually aff ects lower frequencies. SNHL is among the most common sensory impairment aff ecting ~ 17 % of adult Americans. Agerelated hearing impairment (ARHI) or presbycusis impairs 30 % of adults older than 65 years in the US 1 . Heritability and twin studies have suggested genetic contributions to presbycusis and a genomewide-association study recently identifi ed a genetic variant in the metabotropic glutamate receptor 7 ( GRM7 ) gene as a risk factor 2 -5 . However, the majority of the genetic variation underlying susceptibility to SNHL remains unknown. Progressive SNHL in mice displays a histopathology similar to that in humans 6 -9 . Extensive natural variation in mouse strains has caused a remarkable phenotypic and genetic heterogeneity of SNHL, providing experimental model systems to systematically decipher the underlying risk factors 10 .
We recently identifi ed an early-onset hearing loss in Black Swiss (BLSW) mice that is controlled by the quantitative trait loci (QTL) age-related hearing loss 5 ( ahl5 ) and ahl6 (ref. 11), whereby the ahl5 QTL accounts for ~ 60 % of the variation observed. Th e 95 % confidence interval for ahl5 is localized to a 25 Mbp region on chromosome 10 ( D10Mit20 -D10Mit95; 66, 475 -91, 965 kbp). BLSW mice are also sensitive to acoustic stimulation, responding to loud noise with seizures, characterized by wild running, and tonic and clonic convulsions 12 . Sensitivity to audiogenic seizures is highest at 2 -3 weeks of age, but then susceptibility gradually decreases and mice become resistant at 6 weeks of age. Th e underlying locus, juvenile audiogenic monogenic seizure 1 ( jams1 ), localizes to a 1.6cM region that overlaps with the ahl5 interval 12 . Th e genetic location and their related pathology suggested that the ahl5 and jams1 phenotypes are caused by the same allele as previously shown for the 6748delG mutation in the Gpr98 gene causing both audiogenic seizures and hearing loss in the Frings and BUB / BnJ mouse strains 13,14 .
Th e Gipc (GAIP interacting protein, C terminus) genes encode a small family of proteins characterized by a single, centrally located PDZ domain 15 . GIPC1 was fi rst identifi ed through its interaction with regulator of G-protein signalling 19 ( Rgs19 ) and has now been shown to interact with a host of proteins that are involved in signal transduction, vesicular traffi cking, endocytosis and neurotransmitter release 16 -20 . Using its carboxy (C) terminal domain, GIPC1 was demonstrated to interact with Myosin VI, which is associated with SNHL in both mice and humans 21 -23 . Loss of Gipc1 function ( Gipc1 tm1Mvc ) results in a reduction in pre-and postsynaptic transmission in hippocampal neurons 19 .
Here, we identifi ed a sequence polymorphism in the PDZ domain of Gipc3 as the cause of SNHL ( ahl5 ) and audiogenic seizure susceptibility ( jams1 ) in BLSW mice and we implicate mutations in GIPC3 in human recessive SNHL (DFNB15 and DFNB95). Our study suggests a critical role of Gipc3 in signal acquisition and propagation in auditory hair cells.

Results
Positional cloning of ahl5 . To genetically refi ne the ahl5 region, we generated two congenic lines, 10.R and 10.2, by serial backcrossing normal hearing BLSW.CAST-+ / ahl5 progeny to BLSW mice. At generation N8 and higher, there was a signifi cant correlation between segregation of ahl5 alleles and auditory brain stem response (ABR) thresholds ( Fig. 1a ). Recombination events delimited the ahl5 interval to a 2. 19 Mbp region ( Fig. 1b and Supplementary Fig. S1 ). We sequenced 735 of the 831 coding exons in BLSW genomic DNA and identifi ed one nucleotide substitution, which was a guanine to adenine transition in exon 2 of the Gipc3 gene (c.343G > A; Fig. 1c,d ). Th e mutation was absent in 14 inbred and four heterogeneous mouse strains (NMRI, Swiss Webster, CF1 and ICR). However, the 343G / A alleles segregated in mice of the National Institutes of Health (NIH) Swiss founder strain and showed signifi cant correlation with hearing status. Gipc3 343A / A homozygotes had impaired hearing (95 ± 7 dBSPL at 16 kHz), whereas NIH Swiss mice homozygous at the wild-type allele ( Gipc3 343G / G ) exhibited normal hearing (16 ± 5 dBSPL at 16 kHz; Fig. 1e ). Th e missense mutation replaced a glycine with an arginine at position 115 (Gly115Arg) located within the PDZ domain (aa107 -174). Th e Gly115 residue is highly conserved, showing 100 % identity in all published GIPC protein sequences in animals and plants ( Fig. 1f ).
SNHL and audiogenic seizures in BLSW . At 4 weeks of age, BLSW mice showed a moderate hearing impairment with mean thresholds signifi cantly elevated for the click, 8, 16 and 32 kHz stimuli (68 ± 7, 41 ± 6, 41 ± 5 and 85 ± 5 dBSPL) compared with thresholds of 4-weekold normal hearing C3HeB / FeJ mice ( P < 0.001). At 52 weeks of age, BLSW mice exhibited a profound hearing loss that progressed from higher to lower test frequencies. Th e increase in hearing thresholds was most pronounced at 16 kHz with a shift of 48 dBSPL over the threshold of 4-week-old BLSW mice ( Fig. 2a ).
Th e availability of the BLSW.CAST-+ / ahl5 congenic lines allowed us to evaluate the eff ect of ahl5 on the progression of the hearing loss. BLSW.CAST-+ / + mice showed normal hearing at 2 and 12 months of age with mean thresholds of 12 ± 4 and 16 ± 5 dBSPL for the 16 kHz stimulus, respectively ( Fig. 2b ). BLSW.CAST-+ / ahl5 heterozygotes had normal thresholds at 2 months of age (21 ± 6 dBSPL), but exhibited signifi cantly elevated thresholds at 12 months. Th is increase was more prominent in males (57 ± 23 dBSPL) than in females (25 ± 16 dBSPL; P < 0.001, analysis of variance (ANOVA)). Th e threshold increase in 12-month-old female heterozygotes was modest with large standard deviations, but the diff erence compared with 2-month-old females was not signifi cant. Last, the elevated thresholds of male heterozygotes were lower than the thresholds in 12-month-old BLSW mice (89 ± 7 dBSPL). Th ese results suggest that a gender -ahl5 interaction controls the progression of hearing loss in BLSW.CAST-+ / ahl5 heterozygotes. We next measured distortion product otoacoustic emissions (DPOAEs), which are an indicator of outer hair cell (OHC) function. At 4 weeks of age, DPOAEs were reduced at the 5 -20 kHz f2 frequencies (L2 = 65 dBSPL) and were absent between 20 and 55 kHz compared with normal hearing C3HeB / FeJ mice at the same age ( Fig. 2c ). To test for a defect in the stria vascularis, we measured the endocochlear potential (EP). In 10-week-old BLSW mice, the EP was 104 ± 8 mV, which was comparable with potentials obtained from C3HeB / FeJ mice (105 ± 7 mV; Fig. 2d ). Together, the data suggest that BLSW mice exhibit progressive SNHL.
Gipc3 transgenic rescue . To further test that the 343G > A mutation in Gipc3 causes the ahl5 and jams1 phenotypes, we generated transgenic animals expressing Gipc3 . Transgenic mice that were backcrossed to BLSW (N2 -N3) were phenotyped using ABR at 6 -8 weeks of age. In the control groups, Gipc3 343G / A heterozygotes, irrespective of the presence or absence of the transgene (tg( + / − )G / A), expressed normal hearing thresholds ( Fig. 3a and Supplementary Table S1 ) and Gipc3 343A / A homozygotes without the transgene (tg-A / A) exhibited the hearing loss characteristic of the ahl5 phenotype. Interestingly, Gipc3 343A / A homozygotes with the transgene (tg + A / A) showed hearing thresholds to the click, 8 and 16 kHz stimulus that were signifi cantly lower than those of the tg-A / A mice. Furthermore, those thresholds were in the normal range and did not diff er from those of the tg( + / − )G / A heterozygotes ( P > 0.05). For the 32 kHz stimulus tg + A / A mice had thresholds that were elevated compared with tg( + / − )G / A heterozygotes (71 ± 9 dBSPL; P < 0.001), although these thresholds were still lower than those of tg-A / A (92 ± 8 dBSPL). Next, transgenic and non-transgenic N2 -N3 backcross mice were exposed to white noise at 3 weeks of age. Gipc3 343A / A homozygotes without the transgene (tg-A / A) reacted instantly at the 90 dBSPL stimulus with severe convulsions with an average latency period of 24 s. In contrast, Gipc3 343A / A homozygotes with the transgene (tg + A / A) showed a signifi cant delay (108 s); none of the animals seized at the 90 dB stimulus and two animals showed a complete resistance ( Fig. 3b ). Gipc3 343G / A heterozygotes (tg( + / − )G / A) did not show any signs of seizing activity aft er 180 s of stimulation. In conclusion, the Gipc3 transgene rescued the ahl5 hearing defi cit and jams1 audiogenic seizure susceptibility in Gipc3 343A / A homozygotes.
GIPC3 and human recessive deafness . To test whether mutations in GIPC3 also underlie human SNHL, we screened panels of families of Dutch and Indian origin presenting with autosomal recessive nonsyndromic hearing impairment. In one Dutch family, W98-042,    . Blue bars at the bottom of the map represent the congenic segments from CAST / EiJ introgressed onto the BLSW genetic background (red lines). The jams1 locus delimited by the gene Basigin ( Bsg ) and marker D10Mit140 on MMU10 is shown (orange box) 12 . Physical location of Gipc3 within the ahl5 interval is given. Position of the markers is given in kilobase pairs (kbp). ( c ) Gipc3 has six exons and is predicted to encode a 297 amino-acid protein with a central PDZ domain (blue box) fl anked by aminoand C-terminal GIPC homologous domains GH1 and GH2 (grey boxes). Position of the mutation (red annotation) and polyclonal antisera Pb867, Pb869 and Pb877 (green boxes) is shown. ( d ) Sequencing chromatograms demonstrating the 343G > A transition in wild-type (left) and mutant (right) are shown. The nucleotide (A, adenine) and amino acid (Arg, arginine) changes are shown in red. ( e ) ABR thresholds (dBSPL) at click (orange), 8 (green), 16 (purple) and 32 kHz (red) of NIH Swiss homozygous for the wild-type (343G / G; n = 9) and mutant (343A / A; n = 9) Gipc3 alleles. Homozygous (343A / A) NIH Swiss at 6 weeks of age had signifi cantly higher thresholds at all tested stimuli ( P < 0.0001; ANOVA). Data are given as mean ± s.d. ( f ) ClustalW alignment of the amino-acid sequence of Gipc3 homologues and the location of the Gly115Arg relative to the secondary structure, obtained from the GIPC2 crystal structure (DOI:10.2210 / pdb3gge / pdb) is shown. Gly115 maps to a loop (yellow), connecting to β strands, β B (green) and β C (blue).
a homozygous region at 19p13.11-p13.3 encompassing GIPC3 was shared by the two aff ected siblings ( rs11880407 -rs6512152 ; assigned locus symbol: DFNB95). GIPC3 mutation analysis revealed a homozygous nucleotide substitution in exon 6, c.903G > A that segregated with the disease in the family ( Fig. 4a,b and Supplementary  Table S2 ). Th e c.903G > A change is predicted to truncate the protein aft er residue 300 (p.Trp301X) causing the deletion of the C-terminal  In individual II.1, the hearing loss progressed slowly to a loss of 110 dB at 12 years of age and individual II.3, who is now 14 years old, has a stable hearing loss of 90 dB HL ( Fig. 4c ).
In an Indian family, segregating autosomal recessive non-syndromic hearing impairment previous linkage analyses detected a 32 cM region of homozygosity by descent at 19p13 ( D19S209 -D19S411; assigned locus symbol: DFNB15) that included the GIPC3 gene 24 . Sequencing of the six GIPC3 exons identifi ed a homozygous missense mutation, c.785T > G (p.Leu262Arg), in exon 5 that segregated with the deafness in the family ( Fig. 4d,e and Supplementary  Table S3 ). Sequence alignments of GIPC proteins indicated that the Leu262 residue is highly conserved ( Fig. 4f ). Th e mutation was not observed in 161 (322 chromosomes) control individuals.

Hair bundle defects and sensorineural degeneration in BLSW .
Th e high-to-low frequency progression of the hearing loss suggested a histopathology in the organ of Corti. By confocal and scanning electron microscopy we observed an irregular structure of the stereocilia bundle of OHC and inner hair cells (IHC). Starting from postnatal day 3 (P3) onwards, OHC bundles were slightly disoriented and smaller, with bent lateral edges and a rounded apical pole ( Fig. 5a,b ). Th e hair bundle of IHC appeared less rigid and sparse ( Fig. 5a,b ) and at P21 stereocilia were thinner and at times also shorter ( Fig. 5c ). Stereocilia bundles at the base and mid-apical region were present and were aff ected to a similar degree. Histological sections of 8-week-old BLSW ears revealed a normal morphology of the organ of Corti and the spiral ganglion. At 52 weeks of age, there was a signifi cant degeneration of the organ of Corti starting at the base and progressing towards the apex. Concomitantly, the spiral ganglion exhibited a severe loss of neurons that was most obvious at the base and the mid-apical region of the cochlea. Th e organ of Corti and spiral ganglion at the apex were of normal appearance ( Fig. 5d ). Together, these data suggest that Gipc3 is required for postnatal maturation of the hair bundle and long-term survival of hair cells and spiral ganglion.
Reduced Gipc3 protein levels in the BLSW cochlea . We reasoned that the Gly115Arg mutation might alter the expression or stability of Gipc3. To test this hypothesis, we probed western blots with polyclonal antisera raised against Gipc3-specifi c peptides. In protein extracts derived from HEK293T cells transiently expressing wild-type Gipc3 , the Pb867, Pb869 and Pb877 antisera detected a single ~ 40 kDa immunoreactive band ( Fig. 6a and Supplementary Fig. S2 ). When we probed western blots of cochlea and testis protein extracts from BLSW and C3HeB / FeJ we detected a reduction in intensity of the ~ 40 kDa band in BLSW cochlea extracts by 82 ± 7 % ( n = 3) and in testis extracts by 42 ± 9 % ( n = 3) compared with wild-type C3HeB / FeJ extracts ( Fig. 6b ). As an additional test of the expression level diff erences, we generated a mutant Gipc3 343A cDNA construct for expression in HEK293T cells. Quantifi cation, aft er normalizing against actin expression, revealed a reduction in expression levels by 69 % of the mutant Gipc3(115Arg) protein compared with that for wild-type Gipc3(115Gly) ( Fig. 6c,d ). TaqMan qPCR analyses revealed no signifi cant diff erences in Gipc3 mRNA expression in HEK293T cells transfected with wild-type ( Gipc3 343G ) or mutant ( Gipc3 343A ) construct (data not shown). In addition to the ~ 40 kDa band, Pb867 recognizes a ~ 37 kDa band in both cochlea and testis extracts. Both bands can be blocked by the Gipc3-specifi c peptide used for generating the Pb867 antibody and are also detected by the Pb869 / Pb877 antiserum ( Supplementary Fig. S2 ). In addition, Pb867 is specifi c for Gipc3, showing strong reactivity with human GIPC3 and only faint cross reactivity with mouse Gipc1 and Gipc2 ( Supplementary Fig. S2 ). Th us, the ~ 37 kDa band may represent either an alternate isoform or partially cleaved product of the ~ 40 kDa protein. Together, these data indicate that the Gly115Arg substitution causes a reduction of the 40 kDa Gipc3 protein in BLSW cochleae.

Gipc3 localization in hair cells and ganglion neurons .
Using the Pb867 antibody on cochlear sections, we detected Gipc3-specifi c immunoreactivity in IHCs and OHCs, as well as in cochlear spiral ganglion neurons in C3HeB / FeJ mice. Similar expression domains were observed in the BLSW cochlea ( Fig. 6e,f ). Staining was also present in the cytoplasm of vestibular hair cells and vestibular ganglion neurons ( Supplementary Fig. S2 ). On whole-mount organ of Corti preparations, we noted abundant staining in the cytoplasm of IHCs and OHCs characterized by fi ne punctate staining apparent at the base, around the nucleus and throughout the apical part of the hair cells ( Fig. 6g -i ). Th ere was no obvious staining at the stereocilia bundle ( Fig. 6j ). Immunostaining with Pb877 revealed a similar expression and localization profi le, showing staining of the cytoplasm of vestibular and cochlear hair cells and spiral ganglion neurons ( Supplementary Fig. S2 ). Staining with antibodies against Vglut3 and Myosin VI showed localizations similar with Gipc3 in BLSW cochleae ( Fig. 6k,l ). Incubation with nonspecifi c rabbit IgG antiserum produced no staining ( Supplementary Fig. S2 ). Mechanotransduction and K + currents in BLSW . Th e compromised structure of the stereocilia bundle suggested a defect in mechanotransduction. At P6, we found no diff erence in transduction currents recorded from OHCs and IHCs between BLSW and Swiss Webster control mice. Th e mean maximal amplitudes of the transduction currents operating range and adaptation time constants were not signifi cantly diff erent between BLSW and control hair cells. However, by P12 we observed a signifi cant reduction in the maximal transduction current amplitude in both IHCs and OHCs. Th e mean maximal transduction currents recorded from IHCs and OHCs of P12-P14 BLSW mice were reduced by 57 and 71 % , respectively, compared with control hair cells ( Fig. 7a,b ). Th is reduction likely accounts for the elevated hearing thresholds.
To examine voltage-dependent channel activity, we delivered 50-msec voltage steps and measured families of potassium currents from IHCs. At P6, both control and BLSW IHCs had currents with similar amplitudes and kinetics. By P13, control cells had acquired a large rapidly activating potassium current (1726 ± 323 pA at 0 mV) consistent with previous reports 25 . In contrast, potassium currents from IHC of BLSW mice at P12 had signifi cantly smaller amplitudes (869 ± 323 pA at 0 mV; P < 0.01; t -test) not unlike those at P6, albeit with faster activation kinetics ( Fig. 7c ). To consider the functional impact of the attenuated current amplitudes in the BLSW IHCs, we switched to current-clamp mode and delivered current steps of − 100, 100 and 250 pA. Control cells had small membrane potentials consistent with a previous report on wild-type responses 25 , but in BLSW mice the responses were substantially larger, with depolarizations that reached well into the range of calcium channel activation (positive to -60 mV; Fig. 7d ).
ABR amplitudes and audiogenic seizure progression . To investigate the cause of the audiogenic seizure susceptibility and resistance, we correlated various ABR output parameters with seizure progression. Acoustic stimulation showed that BLSW mice were most susceptible to seizures between 2 and 4 weeks of age with mean seizure latencies of 40 ± 42 s ( n = 19) and 89 ± 32 s ( n = 21), respectively. Th ereaft er, latencies increased until animals became completely resistant at 6 weeks of age (180 s, n = 15). Increases in seizure latencies were signifi cant between 3 and 5 weeks and between 4 and 6 weeks of age ( Fig. 8a ).
Last, we correlated ABR thresholds at the click stimulus with seizure progression. ABR thresholds slightly increased in BLSW mice between 2 and 12 weeks of age. However, there were no signifi cant increases in thresholds in mice between 3 (65 ± 6 dBSPL, n = 33) and   5 (67 ± 6 dBSPL, n = 27) and between 4 (69 ± 8 dBSPL, n = 32) and 6 weeks of age (71 ± 7 dBSPL, n = 24; Fig. 8f ). Together, these data suggest that the elevated and subsequent decline of the wave I amplitude correlated with the susceptibility and resistance to audiogenic seizures, respectively.

Discussion
Despite signifi cant advances in the understanding of monogenic deafness, the molecular genetics of the more common and complex forms of hearing loss is poorly characterized. Nine QTLs controlling oligogenic hearing impairment in the mouse have been reported, but, thus far, only two loci have been molecularly characterized ( Cdh23 753A and Fscn2 326A ) 26,27 . Here, we identifi ed the ahl5 / jams1 and DFNB15 / DFNB95 loci uncovering a function for Gipc3 in peripheral auditory signal transmission in mouse and human. Gipc3 distribution in hair cells is reminiscent of vesicular staining and shows a similar localization as Vglut3 and Myosin VI, proteins that regulate synaptic glutamate release and Ca 2 + -dependent exocytosis at the IHC ribbon synapse, respectively 28 -30 . GIPC1 was shown to associate with membranous and cytosolic fractions and also localizes to synaptic vesicles at presynaptic axon terminals of hippocampal neurons 19,31 . Interestingly, glutamate release from presynaptic terminals in the hippocampus was shown to depend on GIPC1 / Myo6 interactions 19 . Given the hair bundle defect, reduced mechanotransduction and K + currents as well as abnormal ABR wave I amplitude in BLSW mice, we hypothesize that Gipc3 may be part of a protein complex involved in vesicle traffi cking that transports factors critical for signal aquisition and propagation in cochlear hair cells.
Th e Gly115Arg change occurring in the loop connecting the β B with the β C strand of the PDZ domain is positioned closely to the peptide recognition pocket 32 . Our western blot data indicate that the Gly115Arg substitution leads to a reduction in Gipc3 protein levels. It is possible that a conformational change or loss of stable interactions may target Gipc3(115Arg) for early degradation. Th e human Trp301X mutation truncates a peptide that shows 42 % sequence identity with the GIPC1 C-terminus. It was recently shown that truncation of 29 residues of the C-terminus of GIPC1 abolishes interaction with Myosin VI suggesting that the full-length C-terminus is required to form stable interactions 33 . In addition, both Leu262 and Trp301 residues are highly conserved among metazoan GIPC proteins. Th ese data indicate that Trp301X and Leu262Arg are pathogenic mutations. It was recently suggested that the transient audiogenic seizures in BLSW mice occur independent of the hearing loss 12 . Indeed, our data do not support a correlation between hearing thresholds and audiogenic seizure progression. Furthermore, the reduced mechanosensitivity seems to be at odds with audiogenic seizure susceptibility. However, our results implicate the ABR wave I amplitude, which represents the summed activity of the cochlear nerve fi bres projecting from IHCs to the cochlear nucleus, in seizure susceptibility and resistance 34 . Th e high wave I amplitude at 2 weeks of age may result from the stunted developmental acquisition of IHC K + currents. Whereas the normal acquisition of potassium currents attenuates the IHC receptor potential, lack of those currents in BLSW allows greater depolarization, which may in turn enhance Ca 2 + -dependent neurotransmission, perhaps leading to synchronized cochlear nerve activity, enhanced ABR wave I amplitudes and seizure susceptibility. Th e subsequent decline in the ABR wave I amplitude and seizure resistance in BLSW mice may be indicative of a neuropathy at the IHC spiral ganglion neuron synapse. In the cochlea, glutamate receptors mediate fast synaptic transmission at the IHC aff erent synapse and glutamate excitotoxicity is central to the neural damage during acoustic trauma 35,36 .
Clinically, the audiogenic seizure pathology in BLSW mice is a form of refl ex epilepsy, in which external sensory stimuli induce seizures. In humans, acoustic refl ex epilepsies, although rare, include startle and musicogenic epilepsies and overt sensitivity to sound also underlies hyperacusis and hyperacute hearing in autism syndrome 37 -40 . Th e pathogeneses underlying these sensitivities are not understood or is it clear whether they originate in the central or peripheral auditory system. BLSW mice and perhaps other mutant alleles of Gipc3 may prove critical to better understand the cause of acoustic hypersensitivity. Our analysis of the hearing loss in BLSW mice has interesting implications for the genetics of human progressive SNHL. GIPC3 and its paralogs are excellent candidate genes for ARHI. It can be argued that ahl5 is a monogenic trait not modelling the degree of genetic complexity presumed to underlie AHRI. Although this argument assumes the common variant-common disease hypothesis, it is also conceivable that rare alleles with strong eff ects in homozygous or heterozygous state may be strong contributors to ARHI. Furthermore, a mutation in the grainyhead like 2 gene ( GRHL2 ) causes progressive hearing loss in humans and a candidate gene approach subsequently associated a GRHL2 variant with ARHI 41,42 . Susceptibility to metabotropic glutamate receptor 7 (mGluR7)-mediated excitotoxicity was suggested as risk factor for ARHI 3 . Interestingly, mGluR7 and Gipc3 show identical localization domains in the mouse cochlea and also share functional similarities 3,18 . Primary neuronal degeneration, which causes hearing impairment because of spiral ganglion degeneration in the absence of hair cell loss, was recently shown to underlie the long-term eff ect of noise exposure 43 . Likewise, hearing loss in BLSW mice at 8 weeks of age occurs without a noticeable hair cell loss. Together, these observations suggest a prominent role for variations in genes aff ecting spiral ganglion function in sensorineural progressive hearing loss.

Methods
Mice and crosses . Black Swiss mice were obtained from Taconic and C3HeB / FeJ mice were purchased from Th e Jackson Laboratory . BLSW mice were originally generated by outcrossing albino NIH Swiss mice to C57BL / 6N and then backcrossing to the NIH Swiss line selecting for black coat colour 44 . In our colony, BLSW mice are maintained by brother × sister mating. To generate BLSW × CAST / EiJ congenic lines (here referred to as BLSW.CAST-+ / ahl5 ), N2 backcross mice 11 that were heterozygous over a 25 Mbp region on chromosome 10 and showed normal hearing thresholds were serially backcrossed to BLSW. At each generation, we assessed the hearing phenotype at 8 weeks of age using ABR threshold measurements at four stimuli (click, 8, 16 and 32 kHz) and genotyped markers located every 10cM along chromosome 10 and markers within the D10Mit20 and D10Mit95 interval.
To generate Gipc3 transgenic mice, a 3292 kbp PaqI DNA fragment of the pBROAD-Gipc3 clone containing the mouse ROSA26 promoter, Gipc3 coding sequence and human β -globin polyadenylation sequence was injected into FVB / Nderived male pronuclei. Transgenic animals were serially backcrossed to BLSW and genotyped by PCR. Institutional review boards at NIH and University of Virginia approved the animal studies.
Auditory tests in mice . For ABR, acoustic stimuli were generated from a high frequency transducer and presented at varying intensity at a rate of 19.1 times per s for a total of 350 presentations. Neuronal responses were captured through subdermal needle electrodes inserted at the vertex, fi ltered, amplifi ed and displayed. Th resholds, latencies and amplitudes were determined using IHS soft ware.
DPOAEs were measured using National Instruments (NI) LabView 8.6 software , operating an NI PCI-4461 Dynamic Signal Analyzer sound card ( National Instruments ), to generate two pure tones, f1 and f2, at the fi xed f2 / f1 ratio of 1.25, which were emitted separately by two Clarion SRU310H high frequency dome tweeters ( Clarion ) placed in the outer ear canal at the presentation level of f2 = f1. Emissions were measured using an Etym õ tic-ER-10B + microphone. Th e amplitude of the 2f1-f2 distortion product was plotted in dBSPL against the f2 frequency where the distortion product is generated.
To measure the EP, a silver / chloride electrode bathed in 0.1 M KCl was inserted through the round window into the endolymph using a remote controlled motorized Micromanipulator ( Piezo World Precision Instrument ). Th e electrode was connected to a Warner Dual Channel Diff erential Electrometer (HiZ-223; Warner Instruments ), which amplifi ed and routed the voltage diff erence (subdermal 1 M KCl reference electrode) to the data acquisition system ( Digidata 1440A , Axon Instruments ) using AxoScope soft ware ( Axon ) at a sampling rate of 10 kHz for 60 s. To induce audiogenic seizures, mice were exposed to white noise of 90, 100 and 110 dBSPL consecutively for 60 s each stimulus. Acoustic stimuli were generated by a Bose loudspeaker ( Bose ) operated by ToneGen soft ware. Speaker levels were calibrated with SoundProDL ( Clarke Analytical Instruments ). Th e mouse was placed in a clear polypropylene box container with a lid with the loud speaker mounted into the side. Each stimulus was maintained for 60 s or until a seizure occurred as evidenced by wild running, fl exing and tonic-clonic convulsions 45 . Seizure latency (sec) was defi ned as the time of the fi rst stimulus onset to the time of seizure onset. Animals resistant to the three consecutive sound stimuli were scored as having a latency of 180 s.
To measure mechanotransduction, the hair bundles were stimulated with a fi re polished glass probe that fi t the shape of the bundles. Th e stimulator was cemented to a PICMA ultra fast piezo actuator ( Physik Instrumente ), driven by a ENV-400 amplifi er ( Piezosystem Jena ). Transduction currents were recorded using the whole-cell, tight-seal technique. Recording pipettes were fi lled with intracellular solution. Membrane potential was clamped to − 64 mV and controlled using an Axopatch Multiclamp 700A amplifi er ( Molecular Devices ). pClamp 8.2 ( Molecular Device ) and Origin 7.5 ( OriginLab ) were used to record and analyse the currents.
Cochleae and testis were homogenized in RIPA buff er containing 1 mM phenylmethylsulphonyl fl uoride and complete protease inhibitor cocktail ( Roche ). Confl uent HEK293 or HeLa cells were lysed on ice in 20 mM Tris-HCl (pH 6.8), 150 mM NaCl, 10 mM EDTA, 1 mM EGTA, 10 mM iodoacetamide, 1 % Triton X-100 with complete protease inhibitor cocktail ( Roche ). For immunoblots, the protein lysate was resolved by 4 -12 % SDS polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, incubated with the primary antibody, complexed with a secondary goat anti-rabbit or goat anti-mouse antibody conjugated to horseradish peroxidase ( Kirkegaard and Perry Laboratories ) and detected using enhanced Chemiluminescence ( Pierce Laboratories ). Th e data were quantifi ed by measuring total protein band intensities ratios using UVP multispectral imaging system and normalized to control α -actin band.
Histology and immunohistochemistry . For gross morphology, the ear was fi xed with 4 % paraformaldehyde, decalcifi ed, dehydrated with a graded series of ethanol, and infi ltrated with JB-4 polymer. Serial sections were cut at 4 μ m thickness and stained with 0.1 % Toluidine Blue O. For cryosections, ears were fi xed, equilibrated in a graded series of sucrose in PBS, transferred to a 2:1 mixture of 20 % Sucrose in PBS:OCT Medium ( Saukra ), frozen over dry ice and ethanol, and sectioned at 12 μ m. For immunostaining of whole-mounts of organ of Corti, cochlea ducts were dissected in Leibovitz and fi xed. Specimens were permeabilized in 0.5 % TritonX-100, incubated with affi nity-purifi ed antibody Pb867 (1:1000) and secondary anti-rabbit IgG (1:1000; Alexa Fluor 594). For visualizing the stereociliary bundle, the specimen was stained with Rhodamine phalloidin ( Invitrogen ) diluted 1:100 in PBS, mounted in ProLong Gold antifade and imaged using a Zeiss LSM confocal microscope. For scanning electron microscopy, ears were fi xed in 4 % glutaraldehyde, 3 mM CaCl 2 , 0.1M HEPES pH 7.4, dissected, washed in PBS, post-fi xed in 1 % OsO 4 , dehydrated in ethanol, critical point dried, sputter coated with platinum and imaged in a Field emission scanning electron microscope ( Hitachi , S-4500).
Plasmid constructs and transfections . Full-length mouse Gipc3 (ref. sequence: NM_148951, GenBank Nucleotide Core) was amplifi ed from CBA / CaJ cochlea cDNA by PCR and cloned into pCR4-TOPO vector ( Invitrogen ) to generate clone pU2. For the transgene construct, a 932-bp Eco RI restriction fragment from plasmid pU2 was cloned into pBROAD3-mcs vector ( InvivoGen ) to generate clone pBroad-Gipc3. For transfection experiments, the 932-bp wild-type cDNA Gipc3 from clone pU2 was cloned in pcDNA3.1( + ) as a Eco RI fragment to create clone pcDNA3.1-Gipc3 . Mutant pcDNA3.1-Gipc3 343A was generated by site-directed mutagenesis using QuickChange mutagenesis kit ( Stratagene ). Constructs were verifi ed by DNA sequencing using the BigDye Terminator v 3.1 following the manufacturer ' s instructions and electrophoresed on a 3730xl capillary sequencer ( Applied Biosystems ). For transient expression, Lipofectamine ( Invitrogen ) was used to transfect HEK293T or HeLa cells with a constant amount of plasmid DNA (1 μ g or 2 μ g) of pcDNA3.1-Gipc3 or pcDNA3.1-Gipc3 343A . Total protein extraction for western blot was performed 48 h aft er transfection.

Molecular genetic analyses in mice .
To sequence coding exons in the ahl5 interval, genomic sequences were obtained from http://genome.ucsc.edu and PCR primers fl anking the exons were designed using Primer3 http://Frodo.wi.mit.edu . Exons were amplifi ed and cycle sequencing was performed as described above. Sequence chromatograms were analysed using Sequencher v4.5 ( Gene Code Corporation ). BLSW DNA sequences were compared against the C57BL / 6J reference sequence. Presence of the Gipc3 343G > A mutation was evaluated by DNA sequencing.
Human patients and mutation analysis . Th e local medical ethics committees at the Radboud University Nijmegen, the Netherlands, the University of Iowa, USA and the University of Madras, India approved the studies. Informed consent was obtained from the participating subject or, in the case of children, from their parents. For the DFNB95 family, members were examined by otoscopy and pure-tone audiometry in a sound-treated room in accordance with current clinical standards. Individual II.1 received a cochlear implant aft er the audiogram taken at 11.5 years of age. High-resolution single-nucleotide polymorphism (SNP) genotyping and mutation analysis was performed as described 46 . Briefl y, genome-wide linkage scans were performed using the Aff ymetrix mapping 250K SNP array ( Aff ymetrix ). Genotypes and regions of homozygosity were defi ned using the Genotyping Console soft ware ( Aff ymetrix ). Primers for amplifi cation of exons and exon -intron boundaries of GIPC3 were designed with ExonPrimer according to the reference sequences NM_133261.2 and NT_011255.14 ( Supplementary Table S2 ). To determine the segregation of the c.903G > A variant in the family and the presence in normal controls, exon 6 was amplifi ed and the PCR product was digested with Sau96I ( New England Biolabs ).
Th e DFNB15 locus segregating in a two-generation consanguineous Indian family with prelingual, profound autosomal recessive hearing impairment was reported previously 24 . Th e GIPC3 gene was amplifi ed using gene-specifi c primers using a standard protocol on a GeneMate Genius thermocycler ( ISC BioExpress ), sequenced and analysed as described above.
Statistical analyses . Unless otherwise indicated groups of data were compared using one-way ANOVA followed by Bonferroni post-tests to correct for multiple testing. GraphPad Prism 4.0b soft ware ( GraphPad ) was used to perform the arithmetic, statistics and to plot the data.