View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Genomics 92 (2008) 219–225 Contents lists available at ScienceDirect Genomics journal homepage: www.elsevier.com/locate/ygeno A locus on distal chromosome 11 (ahl8) and its interaction with Cdh23ahl underlie the early onset, age-related hearing loss of DBA/2J mice Kenneth R. Johnson a,⁎, Chantal Longo-Guess a, Leona H. Gagnon a, Heping Yu b, Qing Yin Zheng b a The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA b Department of Otolaryngology-Head and Neck Surgery, Case Western Reserve University, University Hospitals-Case Medical Center, 11100 Euclid Avenue, Cleveland, OH 44106, USA article info abstract Article history: The DBA/2J inbred strain of mice is used extensively in hearing research, yet little is known about the genetic Received 7 April 2008 basis for its early onset, progressive hearing loss. To map underlying genetic factors we analyzed recombinant Accepted 23 June 2008 inbred strains and linkage backcrosses. Analysis of 213 mice from 31 BXD recombinant inbred strains Available online 15 August 2008 detected linkage of auditory brain-stem response thresholds with a locus on distal chromosome 11, which we designate ahl8. Analysis of 225 N2 mice from a backcross of (C57BL/6J×DBA/2J) F1 hybrids to DBA/2J mice Keywords: confirmed this linkage (LODN50) and refined the ahl8 candidate gene interval. Analysis of 214 mice from a Presbycusis Ahl+ Age-related hearing loss backcross of (B6.CAST-Cdh23 ×DBA/2J) F1 hybrids to DBA/2J mice demonstrated a genetic interaction of Progressive hearing loss Cdh23 with ahl8. We conclude that ahl8 is a major contributor to the hearing loss of DBA/2J mice and that its Inbred strains of mice effects are dependent on the predisposing Cdh23ahl genotype of this strain. DBA © 2008 Elsevier Inc. All rights reserved. Auditory brainstem response Chromosome mapping Quantitative trait loci QTL Complex trait genetics Age-related hearing loss, or presbycusis, negatively affects the quality AHL loci have yet to be identified; however, once identified they can be of life for many elderly individuals [1].Aboutone-thirdofadultsolder tested as candidate genes for human presbycusis susceptibility. than 60 and about half the population by age 80 suffer from a significant DBA/2J is a well-established mouse strain that was derived from hearing loss [2]. Genetic factors have been shown to contribute to the DBA, the oldest of all inbred strains of mice [19]. Mice of the DBA/2J progression and severity of age-related hearing loss (AHL) [3],but strain (henceforth designated D2) begin to exhibit a high-frequency variable environmental factors (such as exposures to loud noise, ototoxic (N16 kHz) hearing loss as early as 3 weeks of age that progresses to a drugs, and disease) coupled with the late onset of the disorder make severe broad frequency hearing loss by 2–3 months and is accom- gene identification studies extremely difficult in human populations [4]. panied by a rapid, progressive degeneration of the organ of Corti and Nonetheless, large-scale association studies of candidate genes have spiral ganglia [20,21]. D2 mice have been used extensively to study begun to identify a few gene variants that may be causative, including various aspects of both peripheral and central hearing loss and related variants of the NAT2 [5], KCNQ4 [6],andGRHL2 genes [7]. pathologies. A PubMed search retrieved more than 100 articles related The laboratory mouse offers a complementary approach for to the use of this strain in auditory research. D2 mice have been used studying the genetic basis and pathophysiology of AHL. Many inbred to study susceptibility to audiogenic seizures and response properties mouse strains exhibit AHL [8] and are easily amenable to genetic and morphologies of auditory neurons [20,22–25]. D2 mice also have analysis [9]. Several quantitative trait loci (QTL) associated with AHL in been used to study the effects of an augmented acoustic environ- mice have been genetically mapped by inbred strain crosses, including ment on the auditory system [21,26]. D2 mice recently were used in a ahl [10,11], ahl2 [12], ahl3 [13,14], ahl4 [15], ahl5 and ahl6 [16], and microarray study of cochlear gene expression, which detected Phl1 and Phl2 [17].Refined genetic and molecular analyses have significant age-related changes in over 4000 genes, including many provided strong evidence that ahl is an inbred strain-specific dimor- that are involved in mitochondrial respiratory chain complexes [27]. phism of the Cdh23 gene [18]. The recessive AHL susceptibility allele Determining the genetic basis of the hearing loss exhibited by D2 mice for this locus is now designated Cdh23ahl and the dominant resistance would aid in the interpretations of these and other auditory studies allele Cdh23Ahl+. The causative gene variants for the other mapped involving this strain. In contrast to the early onset and rapid progression of hearing loss exhibited by D2 mice, high-frequency hearing loss in mice of the ⁎ Corresponding author. Fax: +1 207 288 6149. C57BL/6J inbred strain (henceforth designated B6) is not observed E-mail address: [email protected] (K.R. Johnson). until 3 months of age and does not progress to a broad frequency, 0888-7543/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ygeno.2008.06.007 220 K.R. Johnson et al. / Genomics 92 (2008) 219–225 profound hearing loss until l0 months of age or older [8,28–30]. The Cdh23ahl allele is an important contributor to AHL in many inbred mouse strains, but because B6 and D2 share this susceptibility allele [18], other genetic factors must be responsible for their hearing loss differences. To identify these additional genetic factors, we analyzed recombinant inbred strains and a linkage backcross derived from B6 and D2 parental strains. We detected highly significant linkage of auditory brain-stem response (ABR) thresholds with a locus on distal chromosome (Chr) 11, which we designate ahl8. To evaluate the genetic interaction of ahl8 with Cdh23ahl, we analyzed an independent backcross that segregates alleles at both loci and found that the phenotypic effects of ahl8 genotypes are dependent on a predisposing Cdh23ahl genotype. Results Fig. 1. ABR thresholds of D2, B6, and F1 hybrid mice. D2 mice exhibit an early onset, We assessed hearing in mice by analyzing ABR thresholds to progressive hearing loss as shown by the average ABR thresholds of mice tested at 3.5 broad-band click and pure-tone 8-, 16-, and 32-kHz stimuli [8].We (N=9), 10 (N=5), and 27 (N=7) weeks of age (solid lines). B6D2 F1 hybrids tested at 12 first tested mice of the B6 and D2 parental strains and their F1 hybrids. fi weeks of age (N=10, dashed line) had normal ABR thresholds that were not signi cantly Hearing loss in D2 mice is progressive, with a much earlier onset than different from those of B6 mice (N=15) tested at 30 weeks of age (dotted line). ABR in B6 mice (Fig. 1). For example, at 10 weeks of age the average 16-kHz thresholds are given in dB SPL (decibels, sound pressure level). Standard error bars are shown for each threshold mean. thresholds of D2 mice are about 50 decibels (dB) higher than those of 30-week-old B6 mice, which still retain normal thresholds around 20 dB SPL (sound pressure level). The ABR thresholds of 10 B6D2F1/J Fig. 2. Genetic analysis of hearing loss in BXD recombinant inbred strains. (A) Average 16-kHz ABR thresholds (dB SPL) and their standard errors are shown for 5–12 mice from each of 31 BXD strains (a total of 213 mice) tested at 6–9 months of age. (B) The WebQTL program was used to calculate linkage associations of chromosomal markers with the ABR threshold averages shown in (A). Chromosomes are numbered across the top and megabase positions are given below each chromosome. In a genome-wide scan, only loci on distal Chr 11 exhibited statistically significant associations (LOD=4.3 for D11Mit48). The lower dotted line indicates suggestive (α=0.63) and the upper dotted line indicates significant (α=0.05) genome-wide linkage associations, determined from 1000 permutations. K.R. Johnson et al. / Genomics 92 (2008) 219–225 221 hybrid mice tested at 12 weeks of age were not statistically different from the normal thresholds of 30-week-old B6 mice. ABR thresholds and linkage analysis of BXD recombinant inbred strains As a first approach to genetically mapping loci that underlie the hearing threshold differences between B6 and D2 mice, we analyzed linkage in BXD recombinant inbred (RI) strains. RI lines are useful for obtaining rough estimates of the number of major genes affecting a complex trait and for determining their approximate map locations [31]. The BXD set of RI strains was derived by crossing B6 and D2 mice and then inbreeding separate progeny lines for over 21 generations. We retested 22 of the 25 BXD RI strains that previously had been assessed by Willott and Erway [32] and evaluated 9 new strains [33]. Fig. 4. Refined map position for ahl8 determined from the (B6×D2)×D2 backcross. The In total, we measured ABR thresholds of 213 mice (5–12 mice per 16-kHz ABR thresholds of 225 (B6×D2)×D2 N2 backcross mice were analyzed for strain) from 31 BXD RI strains. The RI strain mice were tested at 6–9 linkage associations with markers on distal Chr 11. Diamonds (13-week test age) and squares (6-week test age) denote lod scores for the following markers: D11Mit285 (89.8 months of age, an age when ABR thresholds of the parental strains Mb), D11Mit360 (103.2 Mb), D11Mit333 (108.6 Mb), D11Mit338 (115.3 Mb), D11Mit48 differ by more than 50 dB (Fig.