Establishment of a Knock-In Mouse Model with the SLC26A4 c.919-2A.G Mutation and Characterization of Its Pathology Ying-Chang Lu1,2., Chen-Chi Wu2,3,4., Wen-Sheng Shen2, Ting-Hua Yang2, Te-Huei Yeh2, Pei-Jer Chen3, I-Shing Yu5, Shu-Wha Lin5, Jau-Min Wong1, Qing Chang6, Xi Lin6, Chuan-Jen Hsu2,7* 1 Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan, 2 Department of Otolaryngology, National Taiwan University Hospital, Taipei, Taiwan, 3 Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan, 4 Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan, 5 Transgenic Mouse Models Core (TMMC), Division of Genomic Medicine, Research Center for Medical Excellence, National Taiwan University, Taipei, Taiwan, 6 Department of Otolaryngology, Emory University School of Medicine, Atlanta, Georgia, United States of America, 7 Department of Otolaryngology, College of Medicine, National Taiwan University, Taipei, Taiwan Abstract Recessive mutations in the SLC26A4 gene are a common cause of hereditary hearing impairment worldwide. Previous studies have demonstrated that different SLC26A4 mutations may have different pathogenetic mechanisms. In the present study, we established a knock-in mouse model (i.e., Slc26a4tm1Dontuh/tm1Dontuh mice) homozygous for the c.919-2A.G mutation, which is a common mutation in East Asians. Mice were then subjected to audiologic assessment, a battery of vestibular evaluations, and inner ear morphological studies. All Slc26a4tm1Dontuh/tm1Dontuh mice revealed profound hearing loss, whereas 46% mice demonstrated pronounced head tilting and circling behaviors. There was a significant difference in the vestibular performance between wild-type and Slc26a4tm1Dontuh/tm1Dontuh mice, especially those exhibiting circling behavior. Inner ear morphological examination of Slc26a4tm1Dontuh/tm1Dontuh mice revealed an enlarged endolymphatic duct, vestibular aqueduct and sac, atrophy of stria vascularis, deformity of otoconia in the vestibular organs, consistent degeneration of cochlear hair cells, and variable degeneration of vestibular hair cells. Audiologic and inner ear morphological features of Slc26a4tm1Dontuh/tm1Dontuh mice were reminiscent of those observed in humans. These features were also similar to those previously reported in both knock-out Slc26a42/2 mice and Slc26a4loop/loop mice with the Slc26a4 p.S408F mutation, albeit the severity of vestibular hair cell degeneration appeared different among the three mouse strains. Citation: Lu Y-C, Wu C-C, Shen W-S, Yang T-H, Yeh T-H, et al. (2011) Establishment of a Knock-In Mouse Model with the SLC26A4 c.919-2A.G Mutation and Characterization of Its Pathology. PLoS ONE 6(7): e22150. doi:10.1371/journal.pone.0022150 Editor: Mai Har Sham, The University of Hong Kong, China Received November 9, 2010; Accepted June 19, 2011; Published July 21, 2011 Copyright: ß 2011 Lu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by research grants from the National Science Council of the Executive Yuan of the Republic of China (No. 96-2314-B-002-174- MY3). The funders had no role in study design, data collection and analysis decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] . These authors contributed equally to this work. Introduction phenotypes observed in Slc26a42/2 mice, such as unanimously profound hearing loss and the absence of thyroid pathology in all Mutations in the SLC26A4 (PDS, GeneID 5172) gene are the littermates, fail to completely reflect those observed in humans second most frequent cause of human hereditary hearing with various SLC26A4 mutations. Clinically, patients with impairment worldwide, following mutations in the GJB2 (GeneID SLC26A4 mutations have been shown to demonstrate a broad- 2706) gene [1]. Recessive SLC26A4 mutations are responsible for spectrum of phenotypes, ranging from non-syndromic isolated both non-syndromic hereditary hearing loss DFNB4 (MIM EVA to full-blown PS with goiter and incomplete partition of the 600791) [2] and Pendred syndrome (PS, MIM 274600) [3], two cochlea in addition to EVA. To date, more than 100 SLC26A4 disorders commonly encountered in patients with deafness. mutations have been identified (Pendred/BOR Homepage; www. DFNB4 and PS are characterized by sensorineural hearing healthcare.uiowa.edu/labs/pendredandbor). It was reported that impairment accompanied by an enlarged vestibular aqueduct patients with different SLC26A4 genotypes were correlated to (EVA, MIM 603545) and/or incomplete partition of the cochlea distinct clinical phenotypes, with PS patients more likely to have 2 (i.e., Mondini dysplasia), although the latter also includes SLC26A4 mutant alleles than those with non-syndromic EVA development of goiter. SLC26A4 encodes pendrin, an iodide/ [9,10]. Furthermore, cell line studies have demonstrated that chloride/bicarbonate transporter expressed in the inner ear, certain SLC26A4 mutations only impair the function of pendrin thyroid, and kidney [4,5,6,7]. partially, instead of completely ablating the protein function In recent years, the understanding of the pathogenesis of [11,12]. These lines of evidence indicate that the pathogenetic DFNB4 and PS has been facilitated by various studies performed mechanisms of SLC26A4 mutations in humans to some extent in the knock-out mouse model Slc26a42/2 [8]. However, the differ from those mechanisms present in the Slc26a42/2 mouse PLoS ONE | www.plosone.org 1 July 2011 | Volume 6 | Issue 7 | e22150 Mouse Model with SLC26A4 c.919-2A.G Mutation model, in which exon 8 of the Slc26a4 gene (GeneID 23985) is mice that did not exhibit circling and 15 Slc26a4tm1Dontuh/tm1Dontuh mice knocked out. that did exhibit circling, were subjected to vestibular evaluations To elucidate the discrepancies in phenotypes observed between (Table 1). In general, Slc26a4tm1Dontuh/tm1Dontuh mice revealed impaired humans and Slc26a42/2 mice, it might be necessary to create balancing ability compared to wild-type mice. The presence of knock-in mouse models that harbor the corresponding human circling behaviors was in complete accord with the presence of head- SLC26A4 mutations. Accordingly, the primary purpose of the tilting in Slc26a4tm1Dontuh/tm1Dontuh mice. Furthermore, circling mice present study was to establish a knock-in mouse model were associated with a significantly poorer performance on reaching, homozygous for the c.919A.G mutation, which is the most swimming, gripping, and rotorod tests. Slc26a4tm1Dontuh/tm1Dontuh mice prevalent SLC26A4 mutation in Chinese [13,14] individuals, and without circling behaviors showed only a minor degree of vestibular the second most prevalent mutation in Japanese [15] and Koreans dysfunction, yet their performance on the rotorod test was also poorer [16]. We also characterized the associated audiological and than wild-type mice. We also noted a significant difference in vestibular phenotypes as well as the inner ear pathology in this vestibular features, including reaching, swimming, gripping, and model. balancing on the rotorod, between Slc26a4tm1Dontuh/tm1Dontuh mice that exhibited circling and those that did not (p,0.05, in all cases). Results Inner ear morphology: endolymphatic duct, vestibular Pathogenetic mechanisms of the c.919-2A.G mutation aqueduct and sac In our previous study, the c.919-2A.G mutation was expected When observed with the naked eye, the orifice of vestibular to lead to aberrant splicing, based on a computer-assisted analysis aqueduct and fovea of endolymphatic sac were noticeably by the Neural Network at the Berkeley Drosophila Genome enlarged in Slc26a4tm1Dontuh/tm1Dontuh mice (Fig. 4B, as indicated Project (BDGP; www.fruitfly.org/seq_tools/splice.html; splice by arrows and dot-dash line), as compared to those in Slc26a4+/+ score = 0 compared to 0.93 of the wild-type sequence [17]. In mice (Fig. 4A). This structural aberrance was also identified with transformed lymphocytes, sequencing of the RT-PCR products revealed that SLC26A4 transcripts from the allele with c.919- histological studies by H&E staining (Fig. 4D vs. Fig. 4C). 2A.G lose the entire exon 8, resulting in fusion of exons 7 and 9 [18]. In the present study, we confirmed that c.919-2A.G indeed Inner ear morphology: cochlea led to a complete omission of exon 8 during the splicing process in Examination of the cochlear duct from 4-week-old mice inner ear and kidney (Fig. 1). The frameshift generated a new stop revealed severe endolymphatic hydrops with dilatation of scala tm1Dontuh/tm1Dontuh codon at position 348, resulting in prematurely truncated pendrin media in Slc26a4 mice (Fig. 5B), compared to +/+ (347 amino acids). However, pendrin localization in the inner ear Slc26a4 mice. A higher magnification view also showed was not affected by the c.919-2A.G mutation, as shown by degeneration of hair cells (Fig. 5B) and a significant atrophy of tm1Dontuh/tm1Dontuh immunofluorescence studies (Fig. 2). the stria vascularis (Fig. 5D) in all Slc26a4 mice by 6 weeks. Degeneration of inner and outer hair cells in the organ of Audiological and vestibular phenotypes Corti could also be confirmed by fluorescence confocal microscopy A total of 45 mice