Mutation at the Evi1 locus in Junbo mice causes susceptibility to otitis media

Abstract

Otitis media (OM), inflammation of the middle ear, remains the most common cause of hearing impairment in children. It is also the most common cause of surgery in children in the developed world. There is evidence from studies of the human population and mouse models that there is a significant genetic component predisposing to OM, yet nothing is known about the underlying genetic pathways involved in humans. We identified an N-ethyl-N-nitrosourea-induced dominant mouse mutant Junbo with hearing loss due to chronic suppurative OM and otorrhea. This develops from acute OM that arises spontaneously in the postnatal period, with the age of onset and early severity dependent on the microbiological status of the mice and their air quality. We have identified the causal mutation, a missense change in the C-terminal zinc finger region of the transcription factor Evi1. This protein is expressed in middle ear basal epithelial cells, fibroblasts, and neutrophil leukocytes at postnatal day 13 and 21 when inflammatory changes are underway. The identification and characterization of the Junbo mutant elaborates a novel role for Evi1 in mammalian disease and implicates a new pathway in genetic predisposition to OM.

Figure 1. The Evi1 Gene Is Mutated in Junbo Mice

(a) Sequence analysis of the Evi1 locus in BALB/c, C3H/HeN, Jbo/+ adult, and Jbo/Jbo embryonic DNA. An A2318T transversion is detected in Jbo/+ and Jbo/Jbo mutants that is not present in either parental substrains. (b) Schematic of the EVI1 peptide. Ten zinc finger motifs are clustered into two DNA-binding domains, ZF1 and ZF2. EVI1 contains a proline-rich repressor domain between the two sets of zinc fingers and a highly acidic domain at the C-terminus. Expanded peptide sequence across the ninth zinc finger motif shows the high degree of conservation of this region between orthologous proteins from different species: Mus musculus (P14404), Rattus norvegicus (ENSRNOG00000012645), Homo sapien (Q03112), Danio rerio (ENSDARP00000008993), Fugu rubrides (ENSDARP00000008993), Drosophila melanogaster (CG31753), Caenorhabditis elegans (R53.3a). Contact residues are highlighted in yellow, the position of the Junbo mutation is highlighted in red. (c) Extra digits are seen on the forelimbs of both heterozygote and homozygote mice E18.5 (white arrows). In heterozygotes, (Jbo/+, middle panel) an extra digit is observed on either forelimb. The homozygotes, (Jbo/Jbo, right panel) have extra digits on both forelimbs. The anterior digit is often reduced in size in the homozygote limbs (red arrow). Wild-type mice, left panel.

Figure 2. Histology of Middle Ear and Nose in Wild-Type and Junbo Mutant Mice

Images (a–h) are from 13-d-old postnatal mice and are given with their original magnification (a) Jbo/+ dorsal section of MEC partly filled with exudate ×40, (b) +/+ normal middle ear temporal bone covered with thin mucoperiosteum (arrowheads) ×400, (c) +/+ inflamed middle ear with thickened mucoperiosteum with neutrophil leukocyte infiltrates and neutrophil-rich exudates in the MEC ×400, (d) Jbo/+ middle ear with more severe suppurative exudation into the MEC ×400, (e) Jbo/+ inflamed edematous polyp (arrowhead) of un-resorbed embryonic middle ear connective tissue, tympanic membrane ×100, (f) Jbo/+ MEC lined by ciliated columnar cells (arrowhead) ×600 (g) Jbo/+ MEC lined by basal cells (arrowhead) ×600, (h) Jbo/+ suppurative rhinitis: nasal cavity with suppurative exudate, nasal septum with inflamed nasal mucosa ×200. Images (i–l) are of adult (180-d) Jbo/+ middle ear with chronic suppurative OM; changes include (i) fibrous polyps (arrowheads) ×200, (j) hyperplasia of ciliated epithelial cells (arrowhead) and fibrosis of mucoperiosteum stroma ×400, and (k) fibrous thickening of the tympanic membrane, outer ear canal ×400 compared with (l) normal +/+ tympanic membrane, outer ear canal ×400. E, exudate; MP, mucoperiosteum; NC, nasal cavity; NM, nasal mucosa; NS, nasal septum; OEC, outer ear canal; TB, temporal bone; TM, tympanic membrane

Figure 3. Histology of the Lung and Middle Ear Exudate in Junbo Mice

(a) 13-d postnatal Jbo/+ lung with perivascular and peribronchiolar cuffs containing Sirius red positive eosinophil leukocytes (arrowhead), bronchiole, pulmonary artery ×400, (b) focal eosiniphilic alveolitis (arrowhead) and thickened alveolar septae (open arrowhead) with eosinophil-rich infiltrates. (c) 21-d postnatal Jbo/+ MEC pus with colonies of Gram positive cocci ×600. B, bronchiole; PA, pulmonary artery

Figure 4. Evi1 Protein Immunostaining in 13-d-Old Jbo/+ and Wild-Type Mice with Acute OM

(a) Jbo/+ positive myeloid cells in temporal bone marrow ×600. Note chondrocytes and osteocytes are also strongly positive. (b) Jbo/+ mucoperiosteum has positive neutrophil leukocytes, fibroblasts, and basal epithelial cell nuclei ×600. (c) +/+ similar pattern of staining in bone marrow ×600 and (d) +/+ inflamed mucoperiosteum ×600. B, basal epithelial cell nuclei; C, chrondrocyte; F, fibroblast; MC, myeloid cell; N, neutrophil leukocyte; O, osteocyte