A new otogelin ENU mouse model for autosomal-recessive nonsyndromic moderate hearing impairment Carole El Hakam, Laetitia Magnol, Véronique Blanquet To cite this version: Carole El Hakam, Laetitia Magnol, Véronique Blanquet. A new otogelin ENU mouse model for autosomal-recessive nonsyndromic moderate hearing impairment. SpringerPlus, SpringerOpen, 2015, 4 (1), pp.2-8. 10.1186/s40064-015-1537-y. hal-02634509 HAL Id: hal-02634509 https://hal.inrae.fr/hal-02634509 Submitted on 27 May 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. El Hakam Kamareddin et al. SpringerPlus (2015) 4:730 DOI 10.1186/s40064-015-1537-y RESEARCH Open Access A new Otogelin ENU mouse model for autosomal‑recessive nonsyndromic moderate hearing impairment Carole El Hakam Kamareddin, Laetitia Magnol and Veronique Blanquet* Abstract Approximately 10 % of the population worldwide suffers from hearing loss (HL) and about 60 % of persons with early onset HL have hereditary hearing loss due to genetic mutations. Highly efficient mutagenesis in mice with the chemical mutagen, ethylnitrosourea (ENU), associated with relevant phenotypic tools represents a powerful approach in producing mouse models for hearing impairment. A benefit of this strategy is to generate alleles to form a series revealing the full spectrum of gene function in vivo. It can also mimic the range of human mutations and polymor- phisms for HL. In the course of a genome ENU mutagenesis program, we selected a new mouse model for hear- ing defect based on a dysmorphological screen. We identified by gene mapping the mutation responsible for this phenotype and characterized it at the histological level of the inner ear and evaluated the vestibule by following the recommendations of the standard operating procedures, IMPReSS. We have identified and characterized a new reces- sive allele of the otogelin gene, Otogvbd/vbd, due to a homozygous one base pair substitution at the splice donor site of intron 29. This mutation leads to a frame-shift and a premature stop codon. We observed a decrease in the amount of sensory cells in the maculae of Otogvbd/vbd mice as well as an apparent drastically decreased density to almost absence of the otoconial membrane. Compared to Otogtm1Prs and twister, the two other existing otogelin alleles, the detailed analysis of Otogvbd/vbd revealed that these mice share some common behavioural characteristics either with Otogtm1Prs or twister whereas the fine vestibular phenotype and the hearing defect are different. Our results emphasize the importance of detecting and characterizing a new allele of a gene in order to get comprehensive information about the gene function. Keywords: Animal model, N-ethyl-N-nitrosourea mutagenesis, Otogelin, Vestibular balance defect, Deafness, Hearing impairement Background sensorineural HI. The conductive HI refers to defects Hearing loss is one of the most common public health in the outer or middle ear. Such a loss yields a mild to issues and comes in the third place for the major moderate HI and it is usually treated with either medi- physical condition after the arthritis and heart dis- cal or surgical intervention. It also indicates a normal ease. About 2–3 of every 1000 children have hearing inner ear activity. In the other hand, sensorineural HI difficulties or deafness (http://www.hearingloss.org). refers to a problem in the inner ear or along the audi- Two types of hearing impairment (HI) are commonly tory pathway; it’s also known as nerve-related hearing known based on the defective part of the hearing loss. In contrast, such loss of hearing yields a mild to organ. They are traditionally classified as conductive or profound HI and could not be completely solved. There is also the mixed HI; it refers to a conductive and a sen- sorineural loss occurring at the same time. While the *Correspondence: [email protected] Univ. Limoges, INRA, UMR 1061, Unité de Génétique Moléculaire conductive component may be treated, the sensorineu- Animale, Faculté des Sciences et Techniques, 123, Avenue Albert Thomas, ral one remains permanent. 87060 Limoges, France © 2015 El Hakam Kamareddin et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 Interna- tional License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Com- mons license, and indicate if changes were made. El Hakam Kamareddin et al. SpringerPlus (2015) 4:730 Page 2 of 8 Hearing loss (HL) can be due to genetic or environ- the acellular membranes covering the six sensory epithe- mental causes or a combination of both. The majority of lial patches of the inner ear (Cohen-Salmon et al. 1997). human hereditary HL (HHL) cases are isolated or asso- In the vestibule, Otog is required for the anchoring of the ciated with only a vestibular dysfunction and so clas- otoconial membrane and the cupula to the neuroepithe- sified as non-syndromic HL (NSHL); but HHL may be lia. In the cochlea, Otog appears to be involved in organ- also accompanied with other abnormalities (syndromic izing the fibrillar network of the tectorial membrane and hearing loss; SHL). More than 50 % of NSHL cases are it likely has a role in determining the resistance of this inherited in an autosomal recessive manner. So far, more membrane to sound stimulation (Simmler et al. 2000a; than 80 loci have been described, and for 60 of these, El-Amraoui et al. 2001). We called the new allele vbd for the mutated genes have been identified (Van Camp and vestibular balance defect. This mouse model displays a Smith 2015 in http://hereditaryhearingloss.org). behavioural and hearing phenotype. This third allele after Model organisms such as the mouse whose genes, twister (twt) and Otogtm1Prs previously characterised in regulatory regions, and genome structure are remark- literature, showed partly characteristics of the two previ- ably similar to those of humans provide powerful tools ous one. Our results highlight the importance of muta- to unravel the functional and evolutionary complexities tions allelic series in the functional analysis of a specific of the human genome (Brown and Hardisty 2003; Oliver gene. et al. 2007; Nguyen and Xu 2008), and so for the under- standing of the molecular mechanisms of hearing. In the Results most of HHL cases, a single mutation in a single gene is Both vestibular and auditory functions are impaired vbd/vbd responsible for HL. In addition, different mutations in in Otog mice the same gene may lead to both syndromic and non-syn- Mutant offspring are clearly identifiable at 4 days of age dromic HHL as well as different onset times which may by their uncontrolled postural response. When placed on vary from birth to old age (http://hereditaryhearingloss. their backs (righting reflex), Otogvbd/vbd mutants stayed org), these occurring human mutations could be more supine without trying to rotate to an upright position closely modelled by mutants identified through ENU- unlike wild-type mice which recovered their posture based screens (Nolan et al. 2002; Aigner et al. 2007). within 10 s. After 2 weeks of age, more than 90 % of Otog- ENU (N-ethyl N-nitrosourea) is one of the most effective vbd/vbd mice had a one side tilted head and showed an alkylating mutagens in mice and it is known to randomly impaired balance. Balance problems were more obvious induce single-point mutations at a rate of ~ 100-fold when animals were held by the tail and dropped onto a higher than the rate of spontaneous mutations (Justice soft surface. Whereas wild-type animals always land on et al. 1999; Balling 2001; Concepcion et al. 2004; Cordes their feet, mutants fell on their back or on their sides. 2005; Gondo 2009). Such chemical mutagenesis induces Such behaviour indicated a saccular defect (Sondag et al. different pedigrees of mice, each one is potentially har- 1998). Mutants showed also abnormal response for the bouring a different DNA mutation for one of many genes elevated platform test. Moreover, about 10 % of Otogvbd/ contained in a chromosome region which are subjected vbd mutants showed a circling behaviour since the post- to a broad range of tests to identify the mutant pheno- natal day 10 (P10) that persists throughout adulthood. type (Hrabé de Angelis et al. 2000; Soewarto et al. 2003; When tested in the swimming test, none of the mutant Davisson et al. 2012). A wide genome screening based mice are able to swim or float properly, but they remain on polymorphic markers followed by DNA sequencing submerged, completely disoriented, while turning under allows the identification of the causal mutation respon- water. They were rapidly removed from water to prevent sible for the detectable phenotype. Furthermore, ENU drowning. This severe vestibular phenotype was fully mutagenesis is particularly a valuable methodology to penetrant. On contrary, wild-type mice came immedi- recover an allelic series of point mutations for any gene ately to the water surface and maintained a horizontal enabling a more acute analysis of gene function (Graw bodyline at the surface. Otogvbd/+ animals responded nor- et al. 2004; Quwailid et al. 2004; Augustin et al. 2005; mally to all the tests. Sachs et al. 2007; Gondo 2009, 2010).