Trombone

an ENU induced mouse model of late-onset deafness

Femke Stelma 1574434

Faculty supervisor: Dr. F.G. Dikkers, Dept of ENT, UMCG Groningen External supervisor: Dr. Mike Bowl, MRC Harwell, Oxfordshire Second external supervisor: Dr. Mahmood Bhutta, Nuffield Dept of Surgical Sciences, University of Oxford Mammalian Genetics Unit, Medical Research Council Harwell, UK

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Summary

Age related hearing loss, or presbycusis, is a growing problem in our ageing society and has profound effects on communication and social interaction. This multifactorial disorder is difficult to study in humans. Mouse models have given us insight in many deafness related genes. Here, a N-ethyl-N-nitrosourea (ENU) induced presbycusis mouse model, Trombone, is presented. Trombone mice show recessive hearing loss that starts in the higher frequencies and is progressive with age. The causative mutation was a T>C change in the gene Slc4a10 which causes a L617P change in the protein, a bicarbonate exchanger. Immunolocalization revealed expression in the type II and V fibrocytes of the spiral ligament, a structure that has a role in endolymph homeostasis. Trombone mice show degeneration of the neighbouring stria vascularis and profound hair cell loss in all cochlear turns. The results suggest an important role for Slc4a10 in hair cell conservation and suggest a novel role for Slc4a10 in the regulation of pH and/or endocochlear potential in the mouse inner ear.

Samenvatting Ouderdomsslechthorendheid, of presbyacusis, is een groeiend probleem in onze vergrijzende samenleving en heeft verstrekkende gevolgen op communicatie en sociale interactie. Deze multifactoriele aandoening is moeilijk te onderzoeken in de mens. Muismodellen hebben ons inzicht gegeven in vele genen die een rol spelen in gehoorsverlies. Een N-ethyl-N-nitrosoureum (ENU) geïnduceerd presbyacusis muismodel, Trombone, wordt hier gepresenteerd. Trombone muizen hebben progressief gehoorsverlies beginnend in de hoge frequenties. De recessieve mutatie, een T>C verandering in het gen Slc4a10 veroorzaakt een L617P verandering in het eiwit, een bicarbonaat transporter. Immunolocalizatie laat expressie zien in type II en V fibrocyten in het ligamentum spirale, een stuctuur betrokken bij de homeostase van endolymfe. Trombone muizen tonen degeneratieve ontwikkelingen van de aan het ligamentum spirale grenzende stria vascularis en tevens haarcel verlies in de gehele cochlea. Deze resultaten laten zien dat Slc4a10 essentieel is voor het behoud van haarcellen en suggereren een rol in de regulatie van pH en/of het endocochleaire potentiaal in het binnen oor.

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Acknowledgements I would like to express my very great appreciation to Dr. Mike Bowl, my supervisor, for his valuable and constructive suggestions during the planning and development of this research project. I would like to offer my special thanks to Prashanthini Shanthakumar, who has been willing to lend me part of her PhD project for 5 months, and who has helped me find my way in the laboratory. I would like to thank Dr. Mahmood Bhutta, who has helped me find this project and was willing guide me as my second supervisor. I would also like to thank my supervisor in the Netherlands, Dr. F.G. Dikkers as well as GIPS-M supervisor professor C.G.M. Kallenberg for their guidance prior and during this project. I am particularly grateful for the assistance given by Andy Parker, who has thought me how to use scanning electron microscopy. Advice given by Jeremy Sanderson has been a great help in using the confocal microscope. Information on immunohistochemical staining and Western Blots provided by Hilda Tateossian was greatly appreciated. I wish to acknowledge the help on bioinformatics provided by Michelle Simon. Finally, I would like to thank the medical faculty of the Rijksuniversiteit Groningen for providing funding that made it possible for me to do this part of my degree in the UK.

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Table of contents Summary/Samenvatting ............................................................................................................ 2

Acknowledgements .................................................................................................................... 3

Table of contents ....................................................................................................................... 4

1. Introduction ........................................................................................................................... 6

1.1 Hearing mechanism ......................................................................................................... 7

1.2 Hearing loss ...................................................................................................................... 8

1.3 Mouse models .................................................................................................................. 8

1.4 Ageing screen MRC Harwell ............................................................................................. 9

1.5 Trombone ......................................................................................................................... 9

2. Methods ............................................................................................................................... 11

2.1 Animal work ................................................................................................................... 11

2.1.1 ENU mutagenesis .................................................................................................... 11

2.1.2 Mating ..................................................................................................................... 11

2.1.3 Matingschemes ....................................................................................................... 12

2.1.4 Mice ........................................................................................................................ 12

2.1.5 Click box and auditory brainstem response (ABR) ................................................. 12

2.2. Genotyping .................................................................................................................... 12

2.2.1 DNA extraction ........................................................................................................ 12

2.2.2 Whole genome mapping......................................................................................... 13

2.2.3 Finemapping using linkage analysis ........................................................................ 13

2.2.4 Mutation detection with NGS Sequencing ............................................................. 14

2.2.5 Validating ENU lesions in candidate region ............................................................ 14

2.2.6 Predicting protein change ....................................................................................... 15

2.3. Phenotyping .................................................................................................................. 15

2.3.1 Histology ................................................................................................................. 15

2.3.2 Measurements on histological sections ................................................................. 16

2.3.3 Immunostaining (DAB) ............................................................................................ 16

2.3.4 Scanning electron microscopy (SEM) sample preparation ..................................... 17

2.4. Statistical analysis ......................................................................................................... 18

3. Results .................................................................................................................................. 19

3.1. ABR data Trombone ...................................................................................................... 19

3.2. Genotyping Trombone mutation .................................................................................. 19

3.2.1 Mapping, single nucleotide polymorphism markers (SNPs) ................................... 19

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3.2.2 Fine mapping ........................................................................................................... 19

3.2.3 Mutation detection with NGS Sequencing ............................................................. 20

3.2.4 Validating ENU lesions in candidate region ............................................................ 20

3.3 Protein prediction Slc4a10 gene product ...................................................................... 21

3.4. Phenotype Trombone ................................................................................................... 22

3.4.1 Weight comparison ................................................................................................. 23

3.4.2 Histology of the inner ear ....................................................................................... 24

3.4.3 Measurements on histological sections ................................................................. 24

3.4.4 Immunohistochemical staining with DAB ............................................................... 25

3.4.5 Ultrastructural studies of the inner ear .................................................................. 25

4. Discussion ............................................................................................................................. 26

4.1 Trombone hearing loss ................................................................................................... 26

4.2 Trombone causative mutation ....................................................................................... 26

4.3 Slc4a10 expression in the inner ear ............................................................................... 28

4.4 Possible function of Slc4a10 in the inner ear ................................................................ 29

4.5 Future work .................................................................................................................... 30

4.6 Clinical implications ....................................................................................................... 31

References ............................................................................................................................... 33

Appendix .................................................................................................................................. 41

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Important abbreviations ABR auditory brainstem response, used for audiometry measurements ENU N-ethyl-N-nitrosourea C3H6N3O2

EP endocochlear potential HET heterozygous HOM homozygous IHC inner hair cell Mb megabases, 1.000.000 basepairs NCBE Na-driven chloride/bicarbonate exchanger (= NBCn2 = Slc4a10) NBCn2 see NCBE NCBT Na- coupled bicarbonate transporters, subgroup in SLC4 family NGS next generation sequencing OHC outer hair cells OTOTO processing fixing method that uses osmium tetroxide and thiocarbohydrazide PCR polymerase chain reaction PDZ protein domain that can bind to other protein SEM scanning electron microscopy SLC4 solute carrier 4 family Slc4a10 gene coding for NCBE protein, mutated in Trombone mice SNP single nucleotide polymorphism

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1. Introduction 1.1 Hearing mechanism

The mechanism of hearing requires highly specialized structures to work closely together. The inner ear is the main player in this process. At this site, in a way that is still not fully understood, sound waves from the surrounding are converted to electrical signals that travel to the brain. Many other parts of the mechanism however, have been unraveled. The main contributors and their function will be discussed briefly. The human ear is composed of three structural compartments, the outer ear, the middle ear and the inner ear, or cochlea (Fig. 1). Through the tympanic membrane and the ossicles in the middle ear, sound waves are conducted from the exterior to the inner ear (Fig. 1A). The inner ear itself is divided in three compartments that run along the length of the cochlea and are filled with fluid (Fig. 1B). The scala media contains endolymph, which is low in Na+ and high in K+ and contributes to the remarkably high potential of +80 mV, known as the endocochlear potential. In contrast, scala vestibule and tympani contain perilymph which is high in Na+ and low in K+. The organ of Corti with the sensory epithelium is located the scala media. The sensory epithelium contains the hair cells, the cells that transduce sound (Fig. 1C). Along the length of the cochlea these hair cells appear as one row of inner hair cells (IHC) and three rows of outer hair cells (OHC)(Fig. 1C).

Figure 1. Mammalian ear. (A) The ear is divided into the inner ear, the middle ear and the outer ear. (B) Cross section of cochlear duct. The cochlea is composed of three compartments; the scala vestibuli, scala media and scala tympani. (C) Organ of Corti with one row of inner hair cells and three rows of outer hair cells (OHC) and surrounding supporting cells. (D) On the apical surface of the OHC stereocilia are arranged in a V-shape and staircase manner. Copied from Dror et al. 2010 (1).

While the IHCs merely function as sensory players, capturing information about frequency, intensity and timing of sound, the OHCs are thought to have an additional function (2). Not only do OHCs transmit information to the cochlear nerve, they also function as cochlear amplifiers, changing their sensitivity and selectivity to sound (3). As sound waves travel through the endolymph, physical vibration will cause the stereocilia on the apex of the hair cells to deflect and move relative to one another. This movement is thought to cause the opening of ion channels which allows an influx of cations from the endolymph, depolarizing the cell; a process known as mechanotransduction. Immediately after depolarization, the hair cell will repolarize and cations will be shifted back into the endolymph, ready to be used again for sound transduction. Different structures are involved in this K+ circulation and homeostasis of the endolymph, including supporting cells and the lateral wall, consisting of the spiral ligament and the stria vascularis.

Hair cells make contact with an afferent neuron. The release of neurotransmitter upon depolarization will excite auditory neurons that signal to auditory centers in the brain and contribute to the perception of hearing.

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1.2 Hearing loss

Age related hearing loss, or presbycusis, is a multifactorial disorder affecting many elderly people worldwide. The prevalence of presbycusis in over-70-year-olds is 64 %, which increases to 84 % in people over 80-years old (4). In our aging society, the number of affected people is likely to increase even further with time. Age related hearing loss is defined by a set of specific characteristics. The hearing loss is bilateral, symmetrical, slowly progressive and will start off in the higher frequencies. As a result, speech recognition will be altered, especially in noisy environments (5). This type of hearing loss therefore has a profound effect on communication and will impair social interaction. Presbycusis is in fact associated with social isolation, depression and reduced quality of life (6,7). Furthermore, presbycusis has been associated with cognitive decline and even dementia, although the specific underlying mechanisms have yet to be elucidated (8). Factors that contribute to age related hearing loss include physiological age-related deterioration of the hearing organ, exposure to loud noise, ototoxic medication, as well as genetic susceptibility. Genetic factors are thought to account for 35 to 55 % of the variance in sensory presbycusis (9). Genes associated with age related hearing loss can be discovered by studying families with presbycusis. For example, by using linkage analysis, over-expression of the tight junction protein TJP2 has been discovered to be a cause of dominant, adult-onset hearing loss (10). Because presbycusis has a late onset phenotype and is thought to be influenced by many different environmental factors, human population studies can be very difficult and time consuming. Therefore, besides studying families with hereditary hearing loss to determine gene function and dysfunction, mice are commonly used as models for hereditary hearing loss. In this way, several genes have been identified that lead to age related hearing loss in mice, including Ahl 1 (Cadherin23)(11), Ahl 4 (citrate synthase)(12), Ahl 5 (Gaip C-terminus-interacting protein 3)(13) and Ahl8 (Fascin-2) (14).

1.3 Mouse models

Due to anatomical, physiological and genetic similarities, mice are powerful tools in unravelling the genetics of many human disorders caused by gene abnormalities, including hereditary hearing loss. The environmental conditions to which the mice are exposed can be closely managed, which is an important factor in age related hearing loss. Furthermore, the mouse genome can be manipulated relatively easy to provide new models for presbycusis. There are a number of possible ways in which mutations can occur in the mouse genome. Frequently used mouse models apply gene targeted mutagenesis like transgenics and reverse genetics to study gene function and generate disease models. These models are genotype driven and target a specific, known gene. A different technique uses the powerful mutagen N-ethyl-N-nitrosourea (ENU)(15). In ENU-mutagenesis, mice are injected with the chemical ENU which causes heritable random point mutations. The resulting mutagenized mice are screened for abnormal phenotypes without knowledge about the causative mutation. When an abnormal phenotype is detected, genetic characterization will be performed to detect the causative gene defect (16).

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The click box emits a high frequency tone (~20 kHz) at a level of 90 dB sound pressure level (SPL). When applied approximately 30 cm above a hearing mouse, the click box will elicit the Preyer reflex in the animal. This reflex consists of a startle response with flicking of the outer ear of the mouse. This response will be diminished or absent in a mouse with profound hearing loss. This relatively gross test is used as a first indication of possible hearing loss.

By analyzing the phenotype in this way, no a priori assumptions are made about the underlying defective gene and its function. As a result, the mouse genome is explored in a non-biased, phenotype-driven way which is likely to uncover novel gene functions. Furthermore, ENU induced point mutations might be a more realistic representation of human disease models than the elimination of the entire gene, as would be done in knockout mice. To date, ENU mutagenesis in mice has contributed to the understanding of many novel disease genes and pathways (17).

1.4 Ageing screen MRC Harwell

At MRC Harwell we use ENU mutagenesis in a large scale project called the Harwell Ageing Mutant Screen. Male mice (G0) are injected with ENU, which causes random point mutations in the DNA of spermatogonial stem cells. After a period of infertility, these mice are mated to a wild type female to produce G1 progeny. The G1 males will all hold different germline ENU mutations they have inherited from their father. These mice will be mated to wild type females to produce different G2. To screen for recessive traits, the G2 female progeny is backcrossed to their G1 father to obtain mice that are homozygous for ENU- induced mutations. The ageing screen consists of a schedule in which the mice undergo systematic recurrent phenotypic screening in different fields, for example behavioural, metabolic, sensory etc. In this way, abnormal mice (phenodeviants) will be identified because they exhibit phenotypic traits that are not observed in their littermates. To screen for hearing loss, click box (see text box) and auditory brainstem response measurements (ABR) are performed. The ABR measurement records the electrical activity in the brain upon an auditory stimulus. The result is a trace consisting of 5 waves that each reflecting a different structure in the auditory pathway. From the data obtained in this way, auditory thresholds can be estimated for different frequencies.

1.5 Trombone The currently examined mouse cohort was a G3 pedigree that was selected from the recessive hearing loss ageing screen. Auditory screening was performed by click box testing and ABR measurements. The ABR thresholds for a subset of animals in this cohort were significantly increased compared to their normal hearing littermates, and therefore it was selected for phenotypic and genotypic characterization. The aim of the study was to discover the genetic basis and molecular origin of hearing loss in this pedigree.

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Figure 1. ABR thresholds of the cohort at the age of 13 months. The affected mice have significantly higher thresholds at all frequencies tested compared to the unaffected mice (p<0.001). Bars show +/- 1 SD.

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8kHz 16kHz 32kHz Click

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Unaffected n=25 Affected n=6

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2. Methods 2.1 Animal work

All the planned animal experiments were reviewed by the MRC Harwell ethics committee before submission of the project for licence of United Kingdom Home Office approval. All animal experiments were carried out under the appropriate licence from the United Kingdom Home Office.

2.1.1 ENU mutagenesis

8–10-Weeks old C57BL/6 male mice were injected with 3 × 100 mg kg−1 body weight ENU, over a 3-week period. Sterility was tested by placing the mice in test-matings for 12 weeks. Mutagenesis was considered to be effective if the mouse was sterile in this period and did not produce any offspring. After the period of sterility, ENU treated mice will regain fertility and be able to produce offspring carrying ENU induced mutations.

2.1.2 Mating

The G0 C57BL/6 male mouse, which was injected with N-ethyl-N-nitrosourea (ENU), was mated to wild type C3H/pde (see text box) females to produce G1 mice. By mating the G0 C57BL/6 male mice to a female of a different strain, known genetic strain-specific SNP variation markers can be used in a later instance for rapid genetic mapping of any ENU induced lesions. As these ENU induced mutations will only be inherited from the father, they will appear on a C57BL/6 background, and not on the maternal C3H/pde background. Genetic analysis of the progeny using known strain-specific SNP variations can differentiate between C57BL/6 and C3H/pde DNA regions and therefore identify a chromosome region likely to hold the causative ENU mutation. A male G1 animal, the founder which will be heterozygous for numerous ENU induced lesions, was outcrossed with a wild type C3H/pde to produce G2 mice (C3H/pde-C57BL/6ENU

G1-Trb/+ x C3H/pde). Female G2Trb/+ animals (1.1) were backcrossed to the G1 male at different times to produce the G3 mice of cohort A. The progeny was screened for abnormal traits (hearing loss) in the ageing screen (Fig. 3). To be able to further investigate the cohort A phenotype, an extra cohort of mice was produced by remating the G1 founder and the female G2Trb/+ animals (1.1) that were used to produce the A cohort. From this progeny, a homozygous affected animal (2.19) was selected and outcrossed to a wildtype C3H/pde animal to produce heterozygous mice on a mixed C3H/pde- C57BL/6 genetic background. The offspring were both backcrossed and intercrossed to produce an additional cohort of G5 Trombone mice (Fig. 3).

C3H/pde is an in house strain which is used as a wild type mouse. Because C3H/HeJ has a retinal phenotype that would interfere with the ageing screen (retinal degeneration 1, RD1 locus, Pde6b) this locus is replaced by the RD1 locus of the BALB/c strain.

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2.1.3 Matingschemes

G0 x C3H/pde G1 (Trb/+) G1 x C3H/pde G2 (1.1/1.2, Trb/+) G1 x G2 (1.1/1.2) G3 Original Cohort A (2.1 – 2.5, Trb/Trb) Extra cohort: G1 x G2 (1.1/1.2) G3 2.19-2.25 (Trb/Trb) G3 (2.19) x C3H/pde G4 3.1/3.2 (Trb/+) G3 (2.19) x G4 (3.1) G5 Trombone cohort (4.1-4.8, Trb/Trb) G4 (3.1) x G4 (3.1) G5 Trombone cohort (5.1-5.5, Trb/Trb)

Figure 3. Schematic representation of matings that were set up to produce Trombone mice. The original cohort A was used in the aging screen. These mice are all aged to 12 months and then culled. To be able to further investigate the phenotype (for example at a set of different time points) additional mice were produced (G5 Trombone cohort). Copied and adjusted from Acevedo-Arozena A, et al. 2008.

2.1.4 Mice

Mice were housed under standard conditions in cages holding up to 5 mice. The mice had free access to water and food at all times.

2.1.5 Click box and auditory brainstem response (ABR)

The mice were subjected to click box (a crude indicator of hearing by observing a startle response upon a high pitch sound) and ABR testing. The ABR test measures the response of the auditory nerve and brainstem to an auditory stimulus. The result is a trace that consists of five peaks (P I-V) each of a different origin. PI; auditory nerve, PII; cochlear nucleus, PIII; superior olivary complex, PIV; the vicinity of the preolivary and lateral lemniscal nuclei and PV; contralateral inferior colliculus. Mice were anaesthetised by intraperitoneal injection of 10 µL Ketaset/Xylazine per gram of bodyweight. (1 ml Ketaset, 0.5 ml Xylazine in 8.35 ml of water) and given regulated auditory stimuli at 1.5 cm from the ear. For each frequency - 8, 16, 32 kHz and click - different sound pressure level stimuli between 0 and 90 dB (5-10 dB steps) were tested. A threshold was defined as the minimal stimulus level that gave a recognizable waveform. The hearing thresholds are displayed in an audiogram. ABR tests were performed at 3, 6, 9 and 13 months. Thresholds in ≥2 frequencies were ≥45 dB were defined as hearing loss. Start of current project, June 2012 (Mainly based on cohort A)

2.2. Genotyping

2.2.1 DNA extraction

Mice were culled by cervical dislocation when reaching the age of 12 months. Inner ears were dissected (see §2.3.5 ‘SEM sample preparation’). DNA was extracted from tail tissue

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with DNeasy Blood & Tissue kit (Qiagen), a silica based DNA purification kit that uses a proteinase-K lysis method.

2.2.2 Whole genome mapping

DNA from 13 (6 affected and 7 unaffected) animals were sent for microarray based whole genome mapping using Illumina GoldenGate Mouse Medium Density (MD) Linkage Panel (Tepnel Life Sciences PLC) that marks C3H/HeJ and C57BL/6. In this way, regions of C57BL/6 homozygosity in affected animals could be identified.

2.2.3 Finemapping using linkage analysis

In order to narrow down the candidate region containing the causative mutation, strain specific SNPs were used which could differentiate between C57BL/6 and C3H/HeJ alleles. Because a SNP is a change in nucleotide sequence in either C57BL/6 or C3H/HeJ DNA, a restriction site could be induced or removed in only one of the two variants. This can then be used to discriminate between C57BL/6 and C3H/HeJ DNA, as one of them will be cut by the restriction enzyme, and the other will not. Primers flanking the SNP are used to amplify the region that holds the change. The amplicon is then digested with the appropriate restriction enzyme. The primers pairs that were designed flanking the SNPs that were used and the corresponding restriction enzyme used for the digest are listed in table 1. Location on Chr 2 (Ensembl

build NCBIM37)

SNP variation Primers Annealing temp (°C)

Product size (bp)

Digest enzyme

51428003 No rs number F AACCAGAAAATTCTCAAAAACTTG 50 250 MwoI R TGCAAAAATGTAGATTAAAATGGTC 53189457 No rs number F TGGATTGGAAGGGACTTGTG 53 246 HaeII R TCCTGATTCTTGTCTCCTCCA 55238068 No rs number F GCCACGTGAAGGATAGCATAA 51 214 NlaIV R TGATAGGGGAAGACCCACTT 56388227 rs3718711 F GGCTCCAGGAAGTGTCTTCCCA 52 271 MfeI HF R AGCTTTCTAAGCCCCAACCTCCTT 63384957 rs50713165 F GGGCACTCGGGTCCTTTA 53 242 MnlI R GGCCATCAAAAGAGTTGCAT 66805958 rs13476553 F TTGCCTGTGGTCATGCTTGTGGG 60 577 BstYI R ACTGCACTAACAGCATGCTCCATGA 69989588 rs4136610 F TGGCAGGCAATGGTGGTCCT 60 577 NlaVI R CACAGTTGATGCATTCACAGTTGAC 74004668 rs29520986 F TGCAGCTTGGCATTATCTTG 57.5 240 NsiI R TTTCTGTGTGTCTGAAGCGAAT 78388769 rs33048145 F GGCATTCCCCTGTACTGAGA 57.5 229 SacI R AATGGCAACAGGCATGTGTA 87353090 rs13476621 F TCCTAGGATGGTCTTGGGACTCA 60 285 BsaJI R TTGGTGTGGTGGGAACCAGC 92826811 rs13476639 F GAGGGGAGGTGGGGAGCAACA 60 408 AciI R GGCAAGGCCCATGGTAGGGA 97160228 rs27363951 F AATCCATTCGCTTAGTGTCCA 57.7 224 ScaI R GGTTTCAGGAGCAGGAACTG 102989561 rs108028077 F GTCCCGTTGCTGCTATGATT 57.7 181 PstI R GGGTGAGTAGGAGCTGTGCT

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PCR was performed with 20 µL ReddyMix PCR Master Mix (Thermo scientific), 0.75 µL (10µM) of each primer (Eurofins) and 2.5 µL 5ng/µL dsDNA per reaction. Reaction: start 95 °C for 3 minutes, 38 cycles of denaturation 94 °C for 20 seconds, annealing between 51 and 60 °C (depending on primer optimum) for 30 seconds and elongation at 72 °C for 30 seconds. Negative controls with omission of dsDNA were included as well as positive controls with C57BL/6, C3H/pde and F1 dsDNA. Post PCR, 10 µL of reaction mix was digested with 0.25 µL restriction enzyme (= 5 Units) (New England Biolabs), 2 µL corresponding NEBuffer (New England Biolabs) and 7.75 µL ddH2O per reaction (if indicated, bovine serum albumin (BSA) (New England Biolabs) was added to the digest mix). The reaction was incubated at the optimal temperature (37 or 60 °C) and time conditions (1 or 3 hours). The digested PCR products were transferred a 2.5% agarose gel for gel electrophoresis separation (150 Volt, 20-30 minutes) and photographed under ultraviolet illumination.

2.2.4 Mutation detection with NGS Sequencing

The whole genome of the founder G1 (C3H/HeJ- C57BL/6ENUF1-Trb/+) male was sequenced at the Wellcome Trust Centre for Human Genetics, Oxford using Illumina HiSeq NGS sequencing. The sequence was aligned to a reference genome by our bioinformatics department, and variations were identified.

2.2.5 Validating ENU lesions in candidate region

Using the NGS data of the G1 founder genome, SNP changes were identified. Our bioinformatics department filtered out the known SNP changes and identified all new changes (ENU mutations) that were not yet known. Of these alleged ENU lesions, the ones in the candidate interval with a quality score above 100 were tested. When the change introduced or removed a restriction site, genotyping was performed by PCR&Digest genotyping as explained above (§2.4). When no restriction site could be used the PCR product was transferred to a 2.5% agarose gel for gel electrophoresis separation and photographed under ultraviolet illumination. The bands of the predicted size were excised from the gel, purified using Geneclean® II Kit (MP Biomedicals) and sent for Sanger sequencing. Location on Chr 2 (Ensembl build

NCBIM37)

Gene containing predicted ENU lesion

Primers Annealing temp. (°C)

Product size (bp)

Digest enzyme

62106906 Slc4a10 F GCAACACTGTGCATCATCCT 52 307 SacI R GGATCAATTCTAGGCCCACTC 64791704 Grb14 F TGTCTGCTTTTCTGCAGGGGA 60 341 - R TTGCCTTTGACAGTTGTTTTGTTGT 69772489 Ubr3 F CATTTATGAGCATGGCTATGGA 55 181 - R AGCCAGGGCTACACAGAGAA

74601679 Hoxd1 F CACGTCCACTATGCCACCT 53 235 -

R TCATCCACTCGAAAGTGCTG

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109758399 Lgr4 F CGGCTGCTCTGCTTCTTC 53 177 - R CACTCACAGTGCTTGGGTGA

2.2.6 Predicting protein change

To investigate whether the alleged ENU induced mutations would have an effect on the protein structure – and therefore might have an effect on its function – the changes of the two high confidence mutations were analyzed using the web tools SIFT, PROVEAN and Polyphen-2. Protein sequence Slc4a10 (62106906 T>C, L617P)

MEIKDQGAQMEPLLPTRNDEEAVVDRGGTRSILKTHFEKEDLEGHRTLFIGVHVPLGGRKSHRRHRHRGHKHRKRDRERDSGLEDGRESPSFDTPSQRVQFILGTEDDDEEHLPHDLFTELDEICWREGEDAEWRETARWLKFEEDVEDGGERWSKPYVATLSLHSLFELRSCILNGTVLLDMHANTIEEIADMVLDQQVSSGQLNEDVRHRVHEALMKQHHHQNQKKLANRIPIVRSFADIGKKQSEPNSMDKNAGQVVSPQSAPACAENKNDVSRENSTVDFSKVDLHFMKKIPPGAEASNILVGELEFLDRTVVAFVRLSPAVLLQGLAEVPIPSRFLFILLGPLGKGQQYHEIGRSIATLMTDEVFHDVAYKAKDRNDLVSGIDEFLDQVTVLPPGEWDPSIRIEPPKNVPSQEKRKIPAVPNGTAAHGEAEPHGGHSGPELQRTGRIFGGLILDIKRKAPFFWSDFRDAFSLQCLASFLFLYCACMSPVITFGGLLGEATEGRISAIESLFGASMTGIAYSLFGGQPLTILGSTGPVLVFEKILFKFCKEYGLSYLSLRASIGLWTATLCIILVATDASSLVCYITRFTEEAFASLICIIFIYEALEKLFELSETYPINMHNDLELLTQYSCNCMEPHSPSNDTLKEWRESNLSASDIIWGNLTVSECRSLHGEYVGRACGHGHPYVPDVLFWSVILFFSTVTMSATLKQFKTSRYFPTKVRSIVSDFAVFLTILCMVLIDYAIGIPSPKLQVPSVFKPTRDDRGWFVTPLGPNPWWTIIAAIIPALLCTILIFMDQQITAVIINRKEHKLKKGCGYHLDLLMVAVMLGVCSIMGLPWFVAATVLSITHVNSLKLESECSAPGEQPKFLGIREQRVTGLMIFILMGSSVFMTSILKFIPMPVLYGVFLYMGASSLKGIQLFDRIKLFWMPAKHQPDFIYLRHVPLRKVHLFTVIQMSCLGLLWIIKVSRAAIVFPMMVLALVFVRKLMDFLFTKRELSWLDDLMPESKKKKLEDAEKEEEQSMLAMEDEGTVQLPLEGHYRDDPSVINISDEMSKTAMWGNLLVTADNSKEKESRFPSKSSPS Protein sequence Grb14 (64791704 A>T, D130E)

MTTSLQDGQSAAGRAGAQDSPLAVQVCRVAQGKGDAQDPAQVPGLHALSPASDATLRGAIDRRKMKDLDVLEKPPIPNPFPELCCSPLTSVLSAGLFPRANSRKKQVIKVYSEDETSRALEVPSDITARDVCQLLILKNHYVDDNSWTLFEHLSHIGLERTVEDHELPTEVLSHWGVEEDNKLYLRKNYAKYEFFKNPMYFFPEHMVSFAAEMNGDRSPTQILQVFLSSSTYPEIHGFLHAKEQGKKSWKKAYFFLRRSGLYFSTKGTSKEPRHLQLFSEFSTSHVYMSLAGKKKHGAPTPYGFCLKPNKAGGPRDLKMLCAEEEQSRTCWVTAIRLLKDGMQLYQNYMHPYQGRSACNSQSMSPMRSVSENSLVAMDFSGEKSRVIDNPTEALSVAVEEGLAWRKKGCLRLGNHGSPSAPSQSSAVNMALHRSQPWFHHRISRDEAQRLIIRQGPVDGVFLVRDSQSNPRTFVLSMSHGQKIKHYQIIPVEDDGELFHTLDDGHTKFTDLIQLVEFYQLNRGVLPCKLKHYCARMAV

2.3. Phenotyping

2.3.1 Histology

To obtain midmodiolar sections of the inner ear, half heads were fixed in formaldehyde. Tissues were embedded in paraffin wax and 5 µm histological sections were cut. Every other section was stained with haematoxylin and eosin according to standard procedures, leaving half of the slides available for immunohistological staining. Pictures of histological sections were taken with an Olympus U-TV0.63XC camera and Olympus CellD software.

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2.3.2 Measurements on histological sections

Measurements on structures in the lateral wall of the cochlea were performed using ImageJ software (http://rsb.info.nih.gov/ij/). The thickness of the stria vascularis was measured in the middle. Surface area measurements of both stria vascularis and spiral ligament were performed (Fig. 4). All measurements were performed three times on the same section to correct for measurement errors and averages were calculated. Surface areas of the stria vascularis were normalized by comparing them to the matching spiral ligament (which had a stable surface area p=0.69). This comparison was done by calculating a stria vascularis : spiral ligament-ratio.

Figure 4. Measurements of the surface area of the stria vascularis and spiral ligament. Measurements were performed in the basal turn of the cochlea of unaffected (A) and affected mice (B). Magnification x 20.

2.3.3 Immunostaining (DAB)

For immunohistological staining a staining method was used including primary, secondary and tertiary antibody. After the sections (5 µm) were dewaxed, endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 30 minutes at room temperature. Non-specific binding of the secondary antibody was blocked by incubating the sections in 10 % rabbit serum for 30 minutes at room temperature. The sections were incubated overnight with 1:800 primary antibody at 4 °C. As negative control the primary antibody was omitted. Nonspecific binding of the primary antibody was tested by blocking the primary antibody (1:200) with immunizing peptide (ratio antibody : blocking peptide = 1:5). After primary incubation, the sections were incubated with biotinylated secondary antibody (1:400) for 30 minutes at room temperature. Subsequently, the slides were incubated with tertiary antibody for 30 minutes at room temperature. A DAB solution was prepared as instructed by manufacturer (liquid DAB+ substrate chromogen system, Dako). The slides were incubated with DAB solution for 1 minute at room temperature. After this, the slides were counterstained with hematoxillin, mounted and photographed.

- Primary antibody: goat anti-NCBE (sc-161917, Santa Cruz Biotechnology, inc.) - Secondary antibody: biotinylated rabbit anti-goat (DAKO).

17

- Tertiary antibody: streptavidin-peroxidase complex (VECTASTAIN elite ABC kit, Vector laboratories)

2.3.4 Scanning electron microscopy (SEM) sample preparation

Mice were culled when reaching the age of 12 months. Inner ears were dissected from wild type and homozygous mice and put in fixative.

Fixation

A 0.1M phosphate buffer was made by diluting 4.44g sodium phosphate monobasic (NaH2PO4) and 17.04g sodium phosphate dibasic (Na2HPO4) in 789ml ddH20. A 2.5 % gluteraldehyde fixative solution was made up by diluting 10 mL 25% gluteraldehyde in 90 mL the 0.1M phosphate buffer (1:10). Mice were anesthetized and culled by bleeding and cervical dislocation. Heads were removed, skinned and bisected down the midline. Inner ears were taken and placed in 5 mL of fixative overnight at 4 °C. Specimens were washed 3 times 15 minutes in 0.1M phosphate buffer and stored at 4 °C. A 4.3% EDTA decalcifying solution was made up by diluting 4.3 g EDTA in 100 mL 0.1 M phosphate buffer. The specimens were decalcified in 1 mL of 4.3% EDTA solution at 4 °C for 48 hours. Specimens were washed 3 times 15 minutes in 0.1M phosphate buffer and stored at 4 °C.

OTOTO processing

Specimens were fine dissected to reveal cochlear hair cells and subsequently treated with osmium tetroxide and thiocarbohydrazide (OTOTO processing). A 1 % solution of osmium tetroxide in 0.1 M sodium cacodylate buffer was made by adding 10 mL of osmium tetroxide to 30 mL of 0.1 M sodium cacodylate buffer. A 1% thiocarbohydrazide solution was made by diluting 0.3 g of thiocarbohydrazide in 30 mL ddH2O. This solution was filtered before use. The samples were fixed in the osmium tetroxide solution at room temperature for 1 hour and washed 6 times for 3 minutes with ddH2O. Next, the samples were placed in the thiocarbohydrazide solution for 30 minutes and washed 6 times for 3 minutes with ddH2O. After this the samples were fixed in the osmium tetroxide solution at room temperature for 1 hour and washed 6 times for 3 minutes with ddH2O again and placed in the thiocarbohydrazide solution for 30 minutes and washed 6 times for 3 minutes with ddH2O again. Finally the samples were fixed in the osmium tetroxide solution at room temperature for 1 hour and washed 6 times for 3 minutes with ddH2O.

Drying & mounting

Samples were dehydrated in ethanol solutions of increasing strength for 45 minutes at 4 °C (25%, 40%, 60%, 80%, 95%), and dehydrated in 100% ethanol for 2x 30 minutes at 4 °C. Samples were dried using critical point drying, mounted in silver paint on SEM stubs and sputter coated with platinum.

Imaging

A JEOL JSM-6010LA InTouchScope™ scanning electron microscope with accompanying software was used to acquire images.

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2.4. Statistical analysis

To compare ABR data (§1.5) as well as measurements on histological sections (§3.1.1) unpaired, two-tailed Students t-tests assuming equal variances were used (Excel 2007). Differences with p-values < 0.05 were considered to be statistically significant.

19

3. Results 3.1. ABR data Trombone The Trombone cohort was identified with significantly elevated hearing thresholds for 16 kHz, 32 kHz and click at 6 months of age. At 9 months of age the thresholds were significantly elevated for 8 kHz, 32 kHz and click. At 13 months of age there was a highly significant difference in all the frequencies between the two groups (Fig. 5). No behavioural defects that could indicate altered vestibular function were detected (i.e. head tossing or circling behaviour), although no vestibular measurements were performed.

Figure 5. ABR thresholds compared between two groups at different ages. Light blue squares, unaffected. Dark blue diamonds, affected; Trombone. *p<0.05, **p<0.01***, p<0.001. Bars show +/- 1 SD.

3.2. Genotyping Trombone mutation

3.2.1 Mapping, single nucleotide polymorphism markers (SNPs)

To find the chromosomal region where the ENU induced mutation was located, the DNA of 13 animals was sent for mapping. The locus of the mutation was initially mapped to a region of approximately 63 megabases (Mb) on chromosome 2 (Chr2:51,321,215- 114,205,619). This region contains 936 genes.

3.2.2 Fine mapping

*** ** *

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Using known SNP markers, the initial 63 megabase (Mb) region was narrowed down to a smaller region of homozygosity of ~22.5 Mb (Chr2:51,428,003 to 74,004,668 = 22,576,665 bases) (Fig. 6).

Figure 6. Fine mapping of candidate region on chromosome 2. (Table) All of the affected animals (red text) are homozygous for C57BL/6 between 51 Mb and 74 Mb. As an example, the two animals at the bottom show that unaffected animals (grey text) are either heterozygous (C57BL/6-C3H/pde) or homozygous for C3H/pde. The figure shows the region of homozygosity on chromosome 2. Red bars: Initial region (63 Mb) and region after narrowing down (22.5 Mb).

3.2.3 Mutation detection with NGS Sequencing

The DNA from the G1 founder male was sent for whole genome sequencing by Illumina Next Generation Sequencing (NGS). NGS allows rapid sequencing of the whole genome. Prior to sequencing, the DNA is divided into many short fragments. In this way, many short pieces of DNA can be sequenced at the same time. Sequencing of these fragments will result in millions of ‘reads’. These reads of ~40-100 bp can subsequently be aligned to a reference genome in order to acquire the sequence of the whole genome (Fig. 7). A higher ‘read depth’ or ‘coverage’ will make the sequencing results more reliable. A change in sequence that is seen in a number of reads will be given a higher quality score, whereas a variant that was only observed in one read will have a low confidence (lower quality score) and a high probability of being a sequencing error. From the NGS data, 86 new SNP changes were identified in the initial 63 MB candidate region.

3.2.4 Validating ENU lesions in candidate region

From these 86 changes, our bioinformatics department identified two high confidence changes which were still located in the candidate region after it was narrowed down (§2.2). Location on Chr2

(Ensembl build NCBIM37) reference change gene amino acid

change

62106906 T C Slc4a10 L617P

64791704 T A Grb14 D130E

Figure 7. Next generation sequencing results for chromosome 2 around 62.000.000 bp area. (A) Sequencing reads aligned to reference genome. Red: forward reads, blue: reverse reads, colours: SNP changes. (B) Magnification of selected area in A. Between dotted lines: single nucleotide polymorphism T>C heterozygous change found at location 62106906 on chromosome 2 in the gene Slc4a10.

Mouse ID SNP 51Mb SNP 53Mb SNP 55Mb SNP 56Mb SNP 63Mb SNP 69Mb SNP 74Mb2.3e B6 B6 B6 B6 B6 B6 B6

2.3i B6 B6 B6 B6 B6 B6 B6

2.5a B6 B6 B6 B6 B6 B6 B6

2.2e Het B6 B6 B6 B6 B6 B6

2.2f Het B6 B6 B6 B6 B6 B6

2.2g Het B6 B6 B6 B6 B6 Het

2.2c C3H Het Het Het Het Het Het

2.3c C3H C3H C3H C3H C3H C3H Het

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The Slc4a10 change introduced a restriction site and could therefore be validated by PCR+digest (Fig. 8). In this way, the change was confirmed to be present in all of the affected animals. The Grb14 change did not introduce a restriction site, so it was amplified and sent for Sanger sequencing. The change was confirmed to be present in one of the affected animals (MPC96/2.5a). Because all affected animals have the same C57BL/6 background in this area, they must all carry this change.

Figure 8. PCR&Digest of Slc4a10. (A) The predicted PCR product with SacI restriction sites. The restriction enzyme SacI cuts the wild type PCR product at two sites resulting in three bands of 40, 97 and 170 bp. The T>C change in Slc4a10 removes one of these restriction sites resulting in only 2 bands of 40 and 267 bp. (B) Result of PCR&Digest. The wild type shows three bands of 40, 97 and 170 bp. The homozygous mutant shows two bands of 40 and 267 bp. The G1 animal is heterozygous for the Slc4a10 mutation and has all 4 bands of 40, 97, 170 and 267 bp. (m) marker, hyperladder IV (NewEnglandBiolabs).

Other lesions with high a quality score were tested (Quality score> 100) Location on Chr2

(Ensembl build NCBIM37) reference change gene amino acid

change

74601679 A C Hoxd1 H174P

69772489 T G Ubr3 V360G

109758399 T G Lgr4 V41G

None of the changes with a quality score above 100 introduced a restriction site and these were therefore by Sanger sequencing. The DNA used came from an affected mouse that was homozygous for the C57BL/6 background across the candidate region (mouseID 2.5a). None of these alleged mutations were present.

3.3 Protein prediction Slc4a10 gene product

Both of the validated high confidence changes were put trough web-based prediction programs SIFT, PROVEAN and Polyphen-2. These programs all look at sequence conservation of the region around the mutation across species and give estimation of the probability that the mutation will affect protein function.

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Program Grb14 D130E

prediction Score Slc4a10 L617P

prediction Score

SIFT tolerated 0.10 affect protein function 0.01 PROVEAN tolerated -1.641 damaging -4.709 PolyPhen-2 benign 0.163 probably damaging 1.00

To investigate the reason this leucine to proline change in the Slc4a10 gene is so likely to affect the protein function of the Slc4a10 gene product, we have to look at the protein sequence and mutation more closely. Figure 9 shows the conservation of the protein sequence for the region around the Slc4a10Trb/Trb mutation.

Figure 9. Sequence conservation of the region around the Trombone Slc4a10 mutation throughout different species. Comparison of the region around the mutation shows high sequence conservation of this region of the Slc4a10 gene on chromosome 2 throughout 12 mammals.

The region is highly conserved throughout different species. This high conservation indicates that this part of the sequence is important for the function of the protein, i.e. no former changes have lasted. Although leucine and proline are both hydrophobic amino acids, their structure is very different. Because proline has an exceptional conformational rigidity compared to other amino acids, it is likely to change the conformation of a protein. Since the gene product of Slc4a10 is a solute carrier built up by multiple transmembrane domains, introduction of a rigid conformational change like this is likely to affect the protein structure and therefore, function.

3.4. Phenotype Trombone Cohort A consisted of 42 animals. The male to female ratio was 22:20. Ten animals died before the endpoint and were excluded from the study (they were either found dead in their cage, died during ABR measurements or were culled because of abnormal weight loss or disease). One of the animals was excluded from the study because it was significantly lighter than the others, mouseID: 2.5b (see §3.4.1). After excluding these animals, the study cohort consisted of 31 animals, the male:female was 17:14. As homozygous females were able to produce normal offspring, the mutation

23

was presumed to have no effect on fertility or development. The litters were normal, there was no fetal death. The gross phenotype of all animals was normal. The ratio deaf: hearing in cohort A was 6:25. This suggests that our mutation inherits in a recessive way. Having found the causative gene, it was possible to re-analyze the ABR-data based on genotype. This strengthened the statement of a recessive mutation, as the hearing abilities of heterozygous animals was no different from that of wild type animals (Fig. 10).

Figure 10. ABR thresholds compared between Slc4a10+/+

, Slc4a10Trb/+

and Slc4a10Trb/Trb

at 13 months. Black triangles, Slc4a10

+/+ light blue squares, Slc4a10

Trb/+, dark blue diamonds, Slc4a10

Trb/Trb. There is no difference

between wild type and heterozygous animals. Homozygous mice have significantly elevated ABR thresholds. ***, p<0.001. Bars show +/- 1 SD.

It is difficult to say anything about the penetrance of the mutation because we do not know which females have littered which progeny (this is due to mating of the mice in trio’s, as a result, we do not know which litter belongs to which mother). All the animals come from the same ancestors. The mice were all housed in the same environment and under the same conditions; therefore unaffected littermates are perfect to act as controls for their affected siblings.

3.4.1 Weight comparison

To determine the influence of the ENU mutation on growth, the weights of affected and unaffected animals at different ages were compared. The data shows that there is no significant difference in body weight course between affected and unaffected animals (Fig.11). N.B. One of the animals (ID 2.5b) seemed to have an abnormal weight course. Because of this, it was excluded from our study data.

Slc4a10Trb/+ n=17

Slc4a10Trb/Trb n=6

Slc4a10+/+ n=8

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Figure 11. Weight course of trombone mice. Animals grew normally. There is no significant difference between weights of affected and unaffected animals, p>0.05. One animal (2.5b) was excluded due to an abnormal weight course. Bars show +/- 1 SD.

3.4.2 Histology of the inner ear

Midmodiolar sections of Slc4a10Trb/Trb animals were compared to those of age- and sex matched wild type or heterozygous littermate controls (Fig. 12). Both wild type and heterozygous (heterozygous data not shown) animals showed no abnormalities of the inner ear. The Slc4a10Trb/Trb inner ears showed a normal organ of Corti and normal outer and inner hair cells. The Reissner’s membrane appeared normal and there was no cell loss in the spiral ganglion neurons and no vacuolization (i.e. cell damage). The stria vascularis appeared to be thinner in the Slc4a10Trb/Trb animals (* in Fig. 12D). To quantify this, surface measurements were performed on the stria vascularis and the spiral ligament (§3.4.3.). Figure 12. Inner ears from Slc4a10

+/+ (A,C,E,F) and Slc4a10

Trb/Trb (B,D,G,H) animals. Compared to the wild

type, the gross morphology of the Slc4a10Trb/Trb

inner ear is normal (A, B). No abnormalities were observed in the organ of Corti or Reissner’s membrane (C,D); The neurons of the spiral ganglion appear normal (E,G). The hair cells in the organ of Corti appear normal (F,H). The stria vascularis appears thinner in the Slc4a10

Trb/Trb

animals (* in D). SG spiral ganglion, RM Reissner’s membrane, TM tectorial membrane, StV stria vascularis, SL spiral ligament, OCorti Organ of Corti, IHC inner hair cell, OHC outer hair cells. Magnifications: A,B x2.5; C, D x20; E-H x100.

3.4.3 Measurements on histological sections

The surface area of the stria vascularis was compared between affected and unaffected mice. To correct for overall size of the cochlea, the surface area was compared to that of the spiral ligament, which had a stable size (p=0.69). The resulting ratio’s are shown in Figure 13. There is a significant difference in strial surface area as well as strial thickness (data not shown) between affected and unaffected mice.

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Figure 13. Comparison between the surface area of the stria vascularis (SV) and the spiral ligament (SL). By comparing the surface area of the SV to the (stable) surface area of the SL insight in the relative size of the SV can be obtained. The decline in ratio in the affected animals shows the lesser contribution of the SV to the lateral wall in this group. ***, p<0.001. Bars show +/- 1 SD.

3.4.4 Immunohistochemical staining with DAB

Slc4a10 expression was compared in cochlea of Slc4a10+/+, Slc4a10+/Trb and Slc4a10Trb/Trb animals. Midmodiolar sections of wild type animals (n=1) as well as heterozygotes (n=2) showed a uniform pattern of expression of Slc4a10 in the spiral ligament. Expression of Slc4a10 was mainly found in type II and V fibrocytes (Fig. 14 A,B). The marginal cells, lining the scala media space do not show expression. No expression was observed in the organ of Corti, Reissner’s membrane, the stria vascularis and other fibrocyte types in the spiral ligament. In Trombone animals (n=5), no immunoreactivity was observed (Fig.14C).

Figure 14. Immunohistochemical staining with Slc4a10 antibody. N.B. Positive DAB staining gives brown colour. Distribution of the Slc4a10 gene product in wild type, Slc4a10

+/+ (A) and heterozygotes Slc4a10

Trb/+ (B)

appears to be confined to type II and V fibrocytes of the spiral ligament (F). In Trombone Slc4a10Trb/Trb

animals (C), no Slc4a10 expression is observed. (D) A negative control without primary antibody prevented staining in wild type, Slc4a10

+/+ animals. (E) Incubation with blocking peptide gave very low background staining in wild

type Slc4a10+/+

animals. Magnification x20. (F) Distribution of different types of fibrocytes (I-V) in spiral ligament.

3.4.5 Ultrastructural studies of the inner ear

Scanning electron microscopy showed that both wild type (n=4) (Fig. 15 A,C,E) and heterozygous (n=11, data not shown) animals have normal hair cells. In Slc4a10Trb/Trb (n=9) significant hair cell loss was observed (Fig. 15 B,D,F). There appears to be more hair cell loss in basal and apical regions of the cochlea than in the middle region. N.B. In the apical region, the wild type hair cells might appear abnormal, but they are in fact normal. The stereocilia in this region are longer and more fragile than further down the cochlea. This makes the stereocilia difficult to fix and conserve their structure.

Figure 15. Scanning electron microscope images of Slc4a10+/+

and Slc4a10Trb/Trb

hair cells. Slc4a10Trb/Trb

animals have significant hair cell loss throughout the whole cochlea. The loss seems to be more severe in the apical and basal regions (B,F) compared to the middle (D). For comparison wild type Slc4a10

+/+ are shown

(A,C,E). Magnification x 2500.

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4. Discussion 4.1 Trombone hearing loss The hearing loss in Trombone mice commences later in life, starting in the high frequencies and is progressive with age, all distinctive features of age related hearing loss. Therefore, the severe phenotype seen at 12 months of age is characterized as the result of presbycusis (5). Whether the hearing loss is also bilateral and symmetrical – two other characteristics of presbycusis – is difficult to say, because per animal only one ear was audiometrically tested. Scanning electron microscopy results indicated that hair cell loss was similar in both ears though, suggesting a symmetrical process.

4.2 Trombone causative mutation The main aim of the study was to discover the genetic basis and molecular origin of the hearing loss in the Trombone animals. In cohort A, no cut-off point between two discovered ENU-induced mutations in Slc4a10 and Grb14 was found. These mutations were both present and either one could therefore theoretically be the cause of hearing loss in Trombone. During the project though, in the extra cohort of G5 Trombone animals that was bred, several affected mice were discovered that were carrying the Slc4a10Trb/Trb mutation, but not the Grb14Trb/Trb mutation. This is consistent with the data we had already found. Unlike the Slc4a10Trb/Trb mutation, the Grb14 Trb/Trb mutation is predicted to be tolerated and will therefore not affect protein function. Furthermore, Grb14 is a growth factor receptor bound protein which has been shown to have an effect on bodyweight; male Grb14-knockout mice have a 5-10% lower bodyweight than wild type controls (18). Comparison of bodyweight in our cohort between affected (hearing loss) animals and wild type mice did not show any significant differences, whilst these animals did carry the Grb14Trb/Trb mutation. Altogether, this data confirms that the ENU-induced Slc4a10Trb allele is the cause of hearing loss in Trombone mice. The change involves a T>C nucleotide substitution which causes a L617P amino acid change in the Slc4a10 gene product, NCBE. Some characteristics of the Slc4a10 gene will be outlined below.

Slc4 family

The solute carrier family 4 (Slc4) contains HCO3- transporters

that play an important role in maintaining intracellular pH. A division can be made between acid loaders and acid extruders (depending on the direction of HCO3

- transport), and electrogenic or electroneutral transporters (depending on stoichiometry). Amongst these transporters are three Na+ independent Cl--HCO3

- exchangers (AE1-3) and five Na+-coupled HCO3

- transporters (NCBT) (Fig. 16) (19). Figure 16. The five NCBTs of the Slc4 family. NCBE = Slc4a10. Copied from Boron, 2009. (19)

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Slc4a10

One of these NCBT’s is the product of the gene Slc4a10, which extrudes acid by transporting bicarbonate into the cell. The Slc4a10 gene product was first cloned by Wang et al. from a mouse insulinoma cell line (MIN6) cDNA library (20). It was initially described as a Na+-driven Cl-/HCO3

-exchanger and therefore designated NCBE (Na-driven chloride/bicarbonate exchanger). Similarly, another group found that pH recovery after an intracellular acid load was dependent on Na+, HCO3

- and Cl- (21). Later, it was proposed that this solute carrier functions as Na+/HCO3

- co-transporter with Cl- self-exchange activity and is not dependant of Cl-. The protein was therefore renamed NBCn2 for Na-bicarbonate co-transporter, electroneutral 2 (22). Recent work however, again proposed that the product of the Slc4a10 gene is in fact a Cl-/HCO3

- exchanger dependant on Na+ and should remain NCBE (23). The reason for this controversy might be the result of differences in study conditions (23). Divergence might also be the result of an actual difference in function between rodents (NCBE) and humans (NBCn2) which has been described before for other proteins of the Slc4 family (24). What all studies agree on, is that the protein transports HCO3

- into the cell, and that this transport is driven by the Na+ transmembrane gradient. In this paper, the Slc4a10 gene product will be referred to as NCBE. Slc4a10 knockout animals have given some insight in the expression and function of NCBE. The net-transport was found to be electroneutral, i.e. resulting in no net charge (22). The Na+:HCO3

- stochiometry is estimated 1:2, with Cl- as counter ion to maintain electroneutrality (23). Different splice variants have been described, depending on alternative splicing of two inserts, insert A and B (Fig. 17).

Figure 17. Construction of the Slc4a10 protein sequence. There are four different splice variants known which depend on the presence or absence of insert A and B. Absence of insert B introduces a PDZ binding motif to the C terminal. Squares represent transmembrane domains (Uniprot prediction). The arrow represents the leucine to proline mutation at location 617, which occurs in a conserved region throughout all four variants.

Of these variants, NCBE-A (without insert A and PDZ motif) seems to be the most abundant in the mouse brain, although distribution of the isoforms varies in different structures (25).

28

The Trombone L>P change occurs between estimated transmembrane domains 4 and 5 and will be present regardless which variant is expressed. Until now, NCBE has mainly been found in the central and peripheral nervous system (21,25-30) and, more recently, in the eye (31). The expression of NCBE in the ear has not been described before. Jacobs 2008 et al., who associated lack of NCBE in the choroid plexus with small brain ventricles, did perform acoustic (click box) startle response on Slc4a10 KO mice, but found no defects (26). In our cohort, only one of the affected mice had a slightly reduced click box response (data not shown), whereas these mice had significantly elevated ABR thresholds. This shows that click box is a crude test and for more accurate results, ABR measurements are needed.

4.3 Slc4a10 expression in the inner ear

Expression of NCBE in wild type, Slc4a10+/+ mice was found in type II and V fibrocytes of the spiral ligament. This expression was lacking in Slc4a10Trb/Trb littermates. Furthermore, the surface area of the stria vascularis in Slc4a10Trb/Trb was significantly reduced. As explained in the introduction, the stria vascularis and spiral ligament in the lateral wall are important players in maintaining endolymphatic homeostasis. This includes controlling [K+] that contributes to the highly positive endocochlear potential (EP) as well as maintaining a stable endocochlear pH (Table 1)(32). Endolymph Perilymph

K+, mM 157 4.2

Na+, mM 1.3 148

Cl-, mM 132 119

HCO3-, mM 31 21

pH 7.5 7.3 Table 1. Fluid composition of endo- and perilymph. Values copied from Wangemann 2007.

The endocochlear potential is generated by transporting high amounts of K+ into the endolymph. Maintaining endocochlear potential is crucial for optimal sound transduction by the sensory hair cells (33). A well regulated pH is important as well, as the functioning of many transport proteins and enzymes depend on optimal pH(32,34). The endolymphatic pH is maintained at a constant level of 7.5 pH. The highly active stria vascularis is a constant source of CO2 production necessitating pH regulation (35). The main buffer used to maintain stable pH is CO2 / HCO3

-. Acidification of the endolymph has been shown to reduce the EP (36). Furthermore, an acidic environment has been associated with an increase in free radical stress in the stria vascularis (37,38). Therefore, disturbance in endocochlear pH homeostasis might result in hearing loss through loss of endocochlear potential and damage by free radical stress.

Participators in acid/base transport

Several transporters that play a role in acid-base regulation in the inner ear have been identified. Apart from Slc4a10, another member of the Slc4 family, Slc4a7, is expressed in a

29

similar pattern in the inner ear. The protein product, NBCn1, is expressed in type I, II and V fibrocytes of the spiral ligament (39). Knock-out mice at 3 months of age show markedly increased ABR thresholds (40). Furthermore, these mice also have a retinal phenotype as was found for Slc4a10 (31,39). Also located in the spiral ligament is K+H+ATP-ase. This enzyme is expressed in many different cell types of the lateral wall. This transporter is proposed to function in K+ circulation, absorbing K+ from the extracellular fluid, as well to contribute to intracellular pH regulation (41). Different subunits of the proton pump (H+-ATPase) are found in the inner ear. H+V-ATP-ase subunit E (V1E) is expressed in the stria vascularis (34). This pump is thought to work together with K+H+antiporters (KHA) forming H+ V-ATP-ase:KHA pair that functions as part of the K+ cycle (42). Mutations in two other H+-ATP-ase subunits, V1B1 (ATP6-V1B1) and V0A4 (ATP6-V0A4), cause hearing loss in combination with renal tubular acidosis (43,44,44-46). Anion exchanger 2, AE2 (Slc4a2) is expressed in the basolateral membrane of epithelial cells facing the endolymphatic compartment and is thought to participate in maintenance of endolymphatic pH (34). Pendrin, the product of gene Slc26a4, is a Na+ independent Cl-/HCO3

- transporter (as well as other anions) and is expressed in epithelial cells of the outer sulcus and spiral prominence cells. This protein is thought to secrete HCO3

- into the endolymph regulating endolymphatic pH. In fact, Pendrin KO mice have acidic endolyphatic pH and are profoundly deaf. Furthermore, these mice display degeneration of the stria vascularis and organ of Corti, a phenotype that resembles the Trombone phenotype (47,48). The strial degeneration in this model is thought to be due to invasion of macrophages, which proposes a possible mechanism for strial degeneration in Trombone mice (49). Other characteristics, like the enlargement of the scala media that is found in Pendrin KO mice (50) are not found in Trombone mice, therefore, additional mechanisms must play a role in the hearing loss in Trombone mice. Figure 18. Schematic representation of the expression of different acid/base transporters. SP; spiral prominence, OS; outer sulcus, RC; root cells.

4.4 Possible function of Slc4a10 in the inner ear

Hearing loss found in mouse models and/or humans lacking the essential acid-base transporters described above, demonstrates the importance of pH regulation in the inner ear. Our findings indicate that Slc4a10 is crucial for normal hearing in mice. The gene product of Slc4a10 is a bicarbonate transporter, NCBE. It is plausible that NCBE is one of the important players of the acid-base regulation within the cochlea.

Acid/base transporters lateral wall. NBCn1 K+H+ATP-ase H+-ATP-ase:KHA pair AE2 Pendrin

30

Being the main structure responsible for generating EP, the stria vascularis is well known to have a high metabolic rate and produce great amounts of CO2 (35). Carbonic anhydrase, which converts the CO2 together with H2O to H+ and HCO3

- is found in the intermediate layer of the stria vascularis, which contains the blood vessels that deliver and remove metabolites (51). But the stria vascularis is not the only site of high metabolism. In the spiral ligament as well, many transporters operating on ATP are found. In fact, the ‘functional fibrocytes’ of the spiral ligament, types II and V, are very rich in mitochondria, indicating high metabolic activity (52). Carbonic anhydrase is also found in the spiral ligament. Although not found by Spicer & Schulte (53) who stained for specific isoforms of CA, Okamura et al − staining more selectively for CA activity − did find CA in type II as well as type V fibrocytes in the spiral ligament (51). Being an acid extruder, NCBE transports HCO3

- into the cell. This transporter could therefore be important for the regulation of intracellular pH in type II and V fibrocytes of the spiral ligament. Indirectly, endolymphatic pH might also be influenced by NCBE bicarbonate transport. Besides transporting HCO3

-, NCBE also transports Cl-. This proposes another function of the protein. NCBE might also work in regulating Cl- concentration, providing the anion that is required for K+ secretion, the main contributor to the generation of EP. The Na+-K+-2Cl- co-transporter Slc12a2 is found in types II and V fibrocytes of the spiral ligament as well as in the stria vascularis and other structures in the inner ear (54). This transporter is essential for uptake of K+ from the intrastrial space. It needs Cl- to function and is part of the K+

circulation, which generates the EP. In aged Slc4a10Trb/Trb mice NCBE is not expressed. This could be the result of apoptosis of fibrocytes, in reaction to the disturbance of intracellular and/or endocochlear homeostasis. Spiral ligament fibrocytes become less abundant towards the apex of the cochlea. For this reason, the apical area is more susceptible to deterioration, which could result in presbycusis (53). The lack of expression can also mean that NCBE fails to target to the plasma membrane. With the protein unable to reach its right location in the cell, it will subsequently be degraded. The fact that Trombone mice have normal hearing early in life suggests that there could be compensation mechanisms taking over the function of NCBE. The progressive nature of the hearing loss in Slc4a10Trb/Trb animals implies that these mechanisms in the spiral ligament could be under stress and fail as one ages. More mouse models have been reported with an association of age related hearing loss with alterations in the lateral wall, like atrophy of the stria vascularis, degeneration of spiral ligament fibrocytes, and loss of certain transporters (55,56). These findings support the hypothesis of Slc4a10 being a novel gene causing recessive presbycusis. To investigate the nature of the Trombone mutation and its effects more extensively more work is needed.

4.5 Future work

Genotype

First of all, to confirm that the causative mutation in Trombone is the one we found in Slc4a10, Slc4a10Trb/Trb mouse should be crossed to a Slc4a10-/- knockout. If Slc4a10Trb/- display hearing loss with the same phenotype as Slc4a10Trb/Trb mice, this will be a confirmation that the mutation in Slc4a10 is the cause of hearing loss in Trombone.

31

Protein

Second, insight in the effects of the mutation could be acquired. By producing tagged constructs, localization of Slc4a10+/+ and Slc4a10Trb/Trb can be investigated. (N.B. During the project this process has been started already, but could not be finished in time) The NBCn2 protein has 11 predicted transmembrane domains (Universal Protein Resource catalog, URL: www.uniprot.org). The trombone mutation is located between TM 4 and TM 5 (L617P). Looking at localization studies can give insight in the reason why there is no expression in Slc4a10Trb/Trbinner ears. A leucine to proline residue change is predicted to introduce an extensive conformational change. This mutation might therefore affect efficient folding and protein localization to the plasma membrane, which could mean that the protein will be degraded.

Phenotype

Trombone animals have age related hearing loss. By looking at the phenotype at different ages, an impression of the development of hearing loss can be obtained. Abundance of other proteins might compensate for the loss of function of NCBE. Immunohistochemical studies have been carried out for NBCn1 (Slc4a7), a protein with very similar function and localization in the inner ear, but the staining was non-specific and therefore did not work. This work should be continued, as abundance of Slc4a7 could indicate a compensatory mechanism. Another possibility is that expression of Slc4a7 is also absent in Trombone, which could indicate loss of fibrocytes. In this case, staining should be performed for apoptotic markers. Since NCBE is a bicarbonate transporter with a probable role in pH regulation, it would be interesting to measure endocochlear pH. As it is likely that EP is affected by pH changes in the endolymph, EP measurements should also be performed. Hilgen et al. have reported a Slc4a10 KO mouse model with visual impairment. (31) Furthermore, Slc4a7, which is expressed in a similar way as Slc4a10 in the inner ear, also has a visiual impaired phenotype (39). By looking at the eye phenotype of Trombone mice, insight can be obtained in the effects of the mutation in the eye. Many acid-base transporters have been identified that not only function in the inner ear, but also in the kidney. The renal function of Trombone mice should therefore be investigated (57).

From mouse to human

Many groups are currently generating genome-wide association study (GWAS) databases, which make it possible to investigate whether a genetic variant (in this case mutations in Slc4a10) is found more often in individuals with a certain phenotype (in this case presbycusis). By collaborating with these groups, an estimation can be made of the possibility that mutations in Slc4a10 are related to human presbycusis.

32

4.6 Clinical implications These results show that Slc4a10 is important for normal functioning of the inner ear. Disruption of Slc4a10 by ENU induced mutagenesis causes age related hearing loss in mice. Little is known about Slc4a10 mutations in humans. A patient with disruption of Slc4a10 through translocation has been described with complex partial epilepsy and mental retardation (58). No report was made of any auditory deficits in this patient. Furthermore, a deletion in Slc4a10 has been detected in a pair of identical twins with autism (59). In the latter report, no information about auditory impairment of patients was described either. Nevertheless, our data indicates that the orthologous human SLC4A10 gene located on chromosome 2 is a candidate for recessive human presbycusis.

33

References

(1) Dror AA, Avraham KB. Hearing loss: mechanisms revealed by genetics and cell biology. Annu Rev Genet 2009;43:411-437.

(2) Steel KP, Kros CJ. A genetic approach to understanding auditory function. Nat Genet 2001 Feb;27(2):143-149.

(3) Brownell WE, Bader CR, Bertrand D, de Ribaupierre Y. Evoked mechanical responses of isolated cochlear outer hair cells. Science 1985 Jan 11;227(4683):194-196.

(4) Pratt SR, Kuller L, Talbott EO, McHugh-Pemu K, Buhari AM, Xu X. Prevalence of hearing loss in Black and White elders: results of the Cardiovascular Health Study. J Speech Lang Hear Res 2009 Aug;52(4):973-989.

(5) Gates GA, Mills JH. Presbycusis. Lancet 2005 Sep 24-30;366(9491):1111-1120.

(6) Ciorba A, Bianchini C, Pelucchi S, Pastore A. The impact of hearing loss on the quality of life of elderly adults. Clin Interv Aging 2012;7:159-163.

(7) Dalton DS, Cruickshanks KJ, Klein BE, Klein R, Wiley TL, Nondahl DM. The impact of hearing loss on quality of life in older adults. Gerontologist 2003 Oct;43(5):661-668.

(8) Lin FR, Ferrucci L, Metter EJ, An Y, Zonderman AB, Resnick SM. Hearing loss and cognition in the Baltimore Longitudinal Study of Aging. Neuropsychology 2011 Nov;25(6):763-770.

(9) Gates GA, Couropmitree NN, Myers RH. Genetic associations in age-related hearing thresholds. Arch Otolaryngol Head Neck Surg 1999 Jun;125(6):654-659.

(10) Walsh T, Pierce SB, Lenz DR, Brownstein Z, Dagan-Rosenfeld O, Shahin H, et al. Genomic duplication and overexpression of TJP2/ZO-2 leads to altered expression of apoptosis genes in progressive nonsyndromic hearing loss DFNA51. Am J Hum Genet 2010 Jul 9;87(1):101-109.

(11) Noben-Trauth K, Zheng QY, Johnson KR. Association of cadherin 23 with polygenic inheritance and genetic modification of sensorineural hearing loss. Nat Genet 2003 Sep;35(1):21-23.

(12) Johnson KR, Gagnon LH, Longo-Guess C, Kane KL. Association of a citrate synthase missense mutation with age-related hearing loss in A/J mice. Neurobiol Aging 2012 Aug;33(8):1720-1729.

(13) Charizopoulou N, Lelli A, Schraders M, Ray K, Hildebrand MS, Ramesh A, et al. Gipc3 mutations associated with audiogenic seizures and sensorineural hearing loss in mouse and human. Nat Commun 2011 Feb 15;2:201.

34

(14) Shin JB, Longo-Guess CM, Gagnon LH, Saylor KW, Dumont RA, Spinelli KJ, et al. The R109H variant of fascin-2, a developmentally regulated actin crosslinker in hair-cell stereocilia, underlies early-onset hearing loss of DBA/2J mice. J Neurosci 2010 Jul 21;30(29):9683-9694.

(15) Acevedo-Arozena A, Wells S, Potter P, Kelly M, Cox RD, Brown SD. ENU mutagenesis, a way forward to understand gene function. Annu Rev Genomics Hum Genet 2008;9:49-69.

(16) Nolan PM, Peters J, Strivens M, Rogers D, Hagan J, Spurr N, et al. A systematic, genome-wide, phenotype-driven mutagenesis programme for gene function studies in the mouse. Nat Genet 2000 Aug;25(4):440-443.

(17) Brown SD, Hardisty-Hughes RE, Mburu P. Quiet as a mouse: dissecting the molecular and genetic basis of hearing. Nat Rev Genet 2008 Apr;9(4):277-290.

(18) Cooney GJ, Lyons RJ, Crew AJ, Jensen TE, Molero JC, Mitchell CJ, et al. Improved glucose homeostasis and enhanced insulin signalling in Grb14-deficient mice. EMBO J 2004 Feb 11;23(3):582-593.

(19) Boron WF, Chen L, Parker MD. Modular structure of sodium-coupled bicarbonate transporters. J Exp Biol 2009 Jun;212(Pt 11):1697-1706.

(20) Wang CZ, Yano H, Nagashima K, Seino S. The Na+-driven Cl-/HCO3- exchanger. Cloning, tissue distribution, and functional characterization. J Biol Chem 2000 Nov 10;275(45):35486-35490.

(21) Giffard RG, Lee YS, Ouyang YB, Murphy SL, Monyer H. Two variants of the rat brain sodium-driven chloride bicarbonate exchanger (NCBE): developmental expression and addition of a PDZ motif. Eur J Neurosci 2003 Dec;18(11):2935-2945.

(22) Parker MD, Musa-Aziz R, Rojas JD, Choi I, Daly CM, Boron WF. Characterization of human SLC4A10 as an electroneutral Na/HCO3 cotransporter (NBCn2) with Cl- self-exchange activity. J Biol Chem 2008 May 9;283(19):12777-12788.

(23) Damkier HH, Aalkjaer C, Praetorius J. Na+-dependent HCO3- import by the slc4a10 gene product involves Cl- export. J Biol Chem 2010 Aug 27;285(35):26998-27007.

(24) Damkier HH, Praetorius J. Genetic ablation of Slc4a10 alters the expression pattern of transporters involved in solute movement in the mouse choroid plexus. Am J Physiol Cell Physiol 2012 May 15;302(10):C1452-9.

(25) Liu Y, Xu JY, Wang DK, Boron WF, Chen LM. Expression and distribution of NBCn2 (Slc4a10) splice variants in mouse brain: cloning of novel variant NBCn2-D. Brain Res 2011 May 16;1390:33-40.

(26) Jacobs S, Ruusuvuori E, Sipila ST, Haapanen A, Damkier HH, Kurth I, et al. Mice with targeted Slc4a10 gene disruption have small brain ventricles and show reduced neuronal excitability. Proc Natl Acad Sci U S A 2008 Jan 8;105(1):311-316.

35

(27) Chen LM, Kelly ML, Rojas JD, Parker MD, Gill HS, Davis BA, et al. Use of a new polyclonal antibody to study the distribution and glycosylation of the sodium-coupled bicarbonate transporter NCBE in rodent brain. Neuroscience 2008 Jan 24;151(2):374-385.

(28) Praetorius J, Nejsum LN, Nielsen S. A SCL4A10 gene product maps selectively to the basolateral plasma membrane of choroid plexus epithelial cells. Am J Physiol Cell Physiol 2004 Mar;286(3):C601-10.

(29) Hubner CA, Hentschke M, Jacobs S, Hermans-Borgmeyer I. Expression of the sodium-driven chloride bicarbonate exchanger NCBE during prenatal mouse development. Gene Expr Patterns 2004 Dec;5(2):219-223.

(30) Chen LM, Choi I, Haddad GG, Boron WF. Chronic continuous hypoxia decreases the expression of SLC4A7 (NBCn1) and SLC4A10 (NCBE) in mouse brain. Am J Physiol Regul Integr Comp Physiol 2007 Dec;293(6):R2412-20.

(31) Hilgen G, Huebner AK, Tanimoto N, Sothilingam V, Seide C, Garrido MG, et al. Lack of the Sodium-Driven Chloride Bicarbonate Exchanger NCBE Impairs Visual Function in the Mouse Retina. PLoS One 2012;7(10):e46155.

(32) Wangemann P. Supporting sensory transduction: cochlear fluid homeostasis and the endocochlear potential. J Physiol 2006 Oct 1;576(Pt 1):11-21.

(33) Hudspeth AJ. How hearing happens. Neuron 1997 Nov;19(5):947-950.

(34) Stankovic KM, Brown D, Alper SL, Adams JC. Localization of pH regulating proteins H+ATPase and Cl-/HCO3- exchanger in the guinea pig inner ear. Hear Res 1997 Dec;114(1-2):21-34.

(35) Marcus DC, Thalmann R, Marcus NY. Respiratory rate and ATP content of stria vascularis of guinea pig in vitro. Laryngoscope 1978 Nov;88(11):1825-1835.

(36) Wangemann P, Nakaya K, Wu T, Maganti RJ, Itza EM, Sanneman JD, et al. Loss of cochlear HCO3- secretion causes deafness via endolymphatic acidification and inhibition of Ca2+ reabsorption in a Pendred syndrome mouse model. Am J Physiol Renal Physiol 2007 May;292(5):F1345-53.

(37) Singh R, Wangemann P. Free radical stress-mediated loss of Kcnj10 protein expression in stria vascularis contributes to deafness in Pendred syndrome mouse model. Am J Physiol Renal Physiol 2008 Jan;294(1):F139-48.

(38) Tanaka F, Whitworth CA, Rybak LP. Round window pH manipulation alters the ototoxicity of systemic cisplatin. Hear Res 2004 Jan;187(1-2):44-50.

(39) Bok D, Galbraith G, Lopez I, Woodruff M, Nusinowitz S, BeltrandelRio H, et al. Blindness and auditory impairment caused by loss of the sodium bicarbonate cotransporter NBC3. Nat Genet 2003 Jul;34(3):313-319.

36

(40) Lopez IA, Acuna D, Galbraith G, Bok D, Ishiyama A, Liu W, et al. Time course of auditory impairment in mice lacking the electroneutral sodium bicarbonate cotransporter NBC3 (slc4a7). Brain Res Dev Brain Res 2005 Nov 7;160(1):63-77.

(41) Shibata T, Hibino H, Doi K, Suzuki T, Hisa Y, Kurachi Y. Gastric type H+,K+-ATPase in the cochlear lateral wall is critically involved in formation of the endocochlear potential. Am J Physiol Cell Physiol 2006 Nov;291(5):C1038-48.

(42) Harvey WR, Xiang MA. K+ pump: from caterpillar midgut to human cochlea. J Insect Physiol 2012 Apr;58(4):590-598.

(43) Vargas-Poussou R, Houillier P, Le Pottier N, Strompf L, Loirat C, Baudouin V, et al. Genetic investigation of autosomal recessive distal renal tubular acidosis: evidence for early sensorineural hearing loss associated with mutations in the ATP6V0A4 gene. J Am Soc Nephrol 2006 May;17(5):1437-1443.

(44) Stover EH, Borthwick KJ, Bavalia C, Eady N, Fritz DM, Rungroj N, et al. Novel ATP6V1B1 and ATP6V0A4 mutations in autosomal recessive distal renal tubular acidosis with new evidence for hearing loss. J Med Genet 2002 Nov;39(11):796-803.

(45) Karet FE, Finberg KE, Nelson RD, Nayir A, Mocan H, Sanjad SA, et al. Mutations in the gene encoding B1 subunit of H+-ATPase cause renal tubular acidosis with sensorineural deafness. Nat Genet 1999 Jan;21(1):84-90.

(46) Lorente-Canovas B, Ingham N, Norgett EE, Golder ZJ, Karet Frankl FE, Steel KP. Mice deficient in the H+-ATPase a4 subunit have severe hearing impairment associated with enlarged endolymphatic compartments within the inner ear. Dis Model Mech 2012 Oct 12.

(47) Everett LA, Belyantseva IA, Noben-Trauth K, Cantos R, Chen A, Thakkar SI, et al. Targeted disruption of mouse Pds provides insight about the inner-ear defects encountered in Pendred syndrome. Hum Mol Genet 2001 Jan 15;10(2):153-161.

(48) Choi BY, Kim HM, Ito T, Lee KY, Li X, Monahan K, et al. Mouse model of enlarged vestibular aqueducts defines temporal requirement of Slc26a4 expression for hearing acquisition. J Clin Invest 2011 Nov;121(11):4516-4525.

(49) Jabba SV, Oelke A, Singh R, Maganti RJ, Fleming S, Wall SM, et al. Macrophage invasion contributes to degeneration of stria vascularis in Pendred syndrome mouse model. BMC Med 2006 Dec 22;4:37.

(50) Kim HM, Wangemann P. Failure of fluid absorption in the endolymphatic sac initiates cochlear enlargement that leads to deafness in mice lacking pendrin expression. PLoS One 2010 Nov 17;5(11):e14041.

(51) Okamura HO, Sugai N, Suzuki K, Ohtani I. Enzyme-histochemical localization of carbonic anhydrase in the inner ear of the guinea pig and several improvements of the technique. Histochem Cell Biol 1996 Oct;106(4):425-430.

37

(52) Garcia Berrocal JR, Mendez-Benegassi I, Marti C, Ramirez Camacho R. Intervention of spiral ligament fibrocytes in the metabolic regulation of the inner ear. Acta Otorrinolaringol Esp 2008 Dec;59(10):494-499.

(53) Spicer SS, Schulte BA. Differentiation of inner ear fibrocytes according to their ion transport related activity. Hear Res 1991 Nov;56(1-2):53-64.

(54) Delpire E, Lu J, England R, Dull C, Thorne T. Deafness and imbalance associated with inactivation of the secretory Na-K-2Cl co-transporter. Nat Genet 1999 Jun;22(2):192-195.

(55) Diaz RC, Vazquez AE, Dou H, Wei D, Cardell EL, Lingrel J, et al. Conservation of hearing by simultaneous mutation of Na,K-ATPase and NKCC1. J Assoc Res Otolaryngol 2007 Dec;8(4):422-434.

(56) Hequembourg S, Liberman MC. Spiral ligament pathology: a major aspect of age-related cochlear degeneration in C57BL/6 mice. J Assoc Res Otolaryngol 2001 Jun;2(2):118-129.

(57) Lang F, Vallon V, Knipper M, Wangemann P. Functional significance of channels and transporters expressed in the inner ear and kidney. Am J Physiol Cell Physiol 2007 Oct;293(4):C1187-208.

(58) Gurnett CA, Veile R, Zempel J, Blackburn L, Lovett M, Bowcock A. Disruption of sodium bicarbonate transporter SLC4A10 in a patient with complex partial epilepsy and mental retardation. Arch Neurol 2008 Apr;65(4):550-553.

(59) Sebat J, Lakshmi B, Malhotra D, Troge J, Lese-Martin C, Walsh T, et al. Strong association of de novo copy number mutations with autism. Science 2007 Apr 20;316(5823):445-449.

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Appendix Appendix Figure 1. Supplementary data of staining with Slc4a10 antibody. N.B. Positive DAB staining gives brown colour. As shown in the result section, distribution of the Slc4a10 gene product in heterozygotes Slc4a10

Trb/+ (A) appears to be confined to type II and V fibrocytes of the spiral ligament (F). In Trombone

Slc4a10Trb/Trb

animals (B-F), no Slc4a10 expression is observed. Magnification x20.

39

Appendix Table 1. ABR data Trombone cohort A.

age, months> 6 months 9 months 12 months

affected ID 8kHz 16kHz 32kHz Click 8kHz 16kHz 32kHz Click 8kHz 16kHz 32kHz Click

2.3e 65 50 65 40 60 40 65 45 70 65 70 65

2.3i 50 65 75 10 60 45 70 25 75 70 100 75

2.5a 55 25 40 50 50 15 70 50 65 60 65 60

2.2e 25 50 45 35 40 15 55 40 80 75 65 75

2.2f 25 40 40 40 10 10 15 10 70 65 60 70

2.2g 40 25 35 25 30 20 55 35 80 70 75 70

average 43.33 42.50 50.00 33.33 41.67 24.17 55.00 34.17 73.33 67.50 72.50 69.17

SD 16.33 15.73 16.12 14.02 19.41 14.63 20.74 14.63 6.06 5.24 14.40 5.85

unaffected ID

2.1a 35 25 25 20 20 30 30 25 30 10 20 15

2.1c 45 25 45 20 15 15 25 30 30 30 10 10

2.1e 40 20 30 25 30 30 25 20 20 15 20 20

2.1f 35 30 40 25 30 10 20 20 15 15 30 20

2.1h 25 30 35 35 25 15 25 10 25 20 15 15

2.1i 40 20 30 30 15 10 35 10 10 15 30

2.1j 25 35 30 15 25 20 30 30 20 20 20 30

2.3a 20 15 40 25 20 20 35 20 15 15 20 10

2.3b 35 25 40 20 15 20 45 20 20 20 20 15

2.3c 45 35 40 25 45 25 25 20 20 25 15 15

2.3d 25 20 30 20 25 15 35 20 50 45 45 25

2.3g 30 25 30 30 35 25 30 25 20 10 20 30

2.3h 35 30 30 25 20 30 35 25 20 25 20 30

2.4a 15 20 25 20 15 20 15 25

2.4c 20 30 40 25 35 25 35 25

2.4g 30 45 50 20 30 25 45 20 20 10 15 10

2.4h 40 35 55 20 25 30 35 25 25 25 25 25

2.2a 25 30 30 30 40 35 20 20 15 25 35 25

2.2b 20 25 30 30 10 30 25 15 20 20 35 25

2.2c 25 30 35 25 40 25 25 15 20 25 25 25

2.2d 50 45 50 25 40 35 50 15 40 35 45 20

2.5e 65 40 65 25 35 30 40 30 20 20 15

2.5g 55 40 50 25 50 40 50 15 45 30 45 20

2.5h 40 40 40 20 30 15 15 30 30 15 25 30

2.5i 35 25 40 30 25 20 15 15 20 30 15 30

average 34.20 29.60 38.20 24.40 27.80 23.80 30.60 21.00 23.91 21.74 24.76 21.30

SD 11.96 8.15 9.99 4.64 10.42 7.94 10.24 5.95 9.77 8.61 10.54 7.11

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Appendix Table 2. Weight data Trombone cohort A.

age, months> 3 6 9 13

affected ID grams grams grams grams

2.3e 28.35 31.12 34.1 34.54

2.3i 30.880 36.76 41.5 47.53

2.5a 29.77 35.69 41.72 40.85

2.2e

40.51 42.76 43.83

2.2f

44.02 46.88 49.71

2.2g

41.3 43.91 44.71

average 29.66667 38.23333 41.81167 43.52833

SD 1.268161 4.632752 4.255953 5.360546

unaffected ID

2.1a 30.12 35.45 49.3 54.69

2.1c 28.37 35.1 42.7 49.16

2.1e 20.64 24.9 26.4 32.66

2.1f 34.58 42.42 44.8 48.02

2.1h 34.74 43.73 48.04 52.79

2.1i 34.28 42.75 45.3 47.93

2.1j 33.32 40.14 44.56 48.7

2.3a 23.45 29.56 35.21 41.7

2.3b 25.46 31 37.94 43.55

2.3c 23.75 33.73 43.68 44.14

2.3d 25.9 39.68 47.42 51.23

2.3g 35.37 48 47.89 50.01

2.3h 30.45 35.4 41.23 44.19

2.4a 25.91 41.89 49.62 54.4

2.4c 25.79 32.49 37.35 42.66

2.2a 25.91 34.32 37.83 41.5

2.2b 29.96 39.39 43.56 44.63

2.2c 26.08 34.22 42.88 46.51

2.4g 38.08 45.61 51.51 49.18

2.4h 31.23 35.94 42.94 43.75

2.5e 32.18 38.19 38.04 37.58

2.5g

34.41 37.81 37.85

2.5h

36.3 37.59 40.85

2.5i

47.22 52.11 55.22

2.2d 34.82 45.38 49.85 52.16

average 29.56318 37.8888 43.0224 46.2024

SD 4.723932 5.796808 6.02416 5.789238

41

Appendix Table 3. Measurements of stria vascularis and spiral ligament on histological sections.

Stria Stria SL+stria SL ratio ID thickness surface surface surface Stria/SL um um2 um2 um2 WT 17.586 4529.132 42504.815 37975.683 2.19g 17.586 4560.124 41837.789 37277.665 17.618 4431.612 42041.511 37609.899 17.59667 4506.96 42128.04 37621.08 0.12 HET 20 4190.025 37552.893 33362.868 4.2f 20.208 3873.648 38693.388 34819.74 18.328 3866.58 38086.777 34220.197 19 3976.751 38111.02 34134.27 0.12 HET 20.42 4433.626 45732.985 41299.359 2.20b 18.35 4334.229 45566.842 41232.613 18.18 4453.136 45781.462 41328.326 18.98 4406.997 45693.763 41286.766 0.11 Het 15.307 2598.161 36365.518 33767.357 4.2c 14.903 2761.13 36181.761 33420.631 14.703 2754.063 35535.703 32781.64 14.971 2704.451 36027.661 33323.21 0.08 HOM 13.457 2630.579 43514.565 40883.986 2.20a 15.26 2557.231 43921.989 41364.758 14.746 2411.777 44691.106 42279.329 14.48767 2533.196 44042.553 41509.35767 0.06 HOM 14.903 2539.749 36936.773 34397.024 4.2a 14.192 2071.419 36787.823 34716.404 15.198 2273.468 37037.338 34763.87 14.76433 2294.879 36920.645 34625.766 0.07 HOM 13.307 2704.382 37655.14 34950.758 4.2d 12.182 2798.131 37785.682 34987.551 13.388 2528.306 38748.533 36220.227 12.959 2676.94 38063.118 35386.17867 0.08 HOM 13.152 3004.345 40470.058 37465.713 2.19h 14.363 2948.686 42006.188 39057.502 12.71 2956.047 41388.45 38432.403 13.41 2969.69 41288.23 38318.54 0.08