Elmod1 Mutations Cause Inner Ear Dysfunction Figure 4. Molecular analysis of the Elmod1rda mutation. Exons 1–5 of Elmod1 are deleted in rda mutant chromosomes. A. Southern blots of genomic DNA from mice with +/+ (lanes marked 1), +/rda (lanes marked 2) and rda/rda (lanes marked 3) genotypes, digested with BamHI (Bam), EcoRI (Eco), PstI (Pst), and PvuII (Pvu) restriction enzymes. The blots shown were hybridized with Elmod1 cDNA probes corresponding to exons 3–7 (nucleotides 426–904 of NM_177769, blot on left) and exons 3–4 (nucleotides of NM_177769,blot on right). Elmod1 mRNA is not expressed in rda/rda mutant mice. B. Northern blots of total RNA extracted from brains of adult C57BL/6J controls (+/+) and rda/rda mutants was hybridized first with an Elmod1 cDNA probe (nucleotides 426–904 of NM_177769, top figure) and subsequently with a beta actin control probe (Actb, bottom) to evaluate RNA loading concentrations. C. PCR with primer pairs spanning the genomic region around Elmod1 was used to define the extent of the rda deletion. Primers immediately flanking the mutation amplified a 963 bp product in mutant (rda) but not wildtype (+) DNA, and sequencing of this product identified a 757 bp insertion. A three-primer assay was developed to distinguish homozygous wildtype mice (+), rda/+ heterozygotes (het), and rda/ rda homozygous mutants (rda). D. The molecular structure of the rda mutation was precisely defined as a 137,786 bp deletion with a 757 bp inverted insertion at the site of the deletion. Exons 1–5 of Elmod1 and the entire Tnfaip8l3 gene are included in the deletion. doi:10.1371/journal.pone.0036074.g004 detected in retinal ganglion cells of the eye examined at P2 (Figure S2C) and at P15 (not shown). Although Elmod1 appears to be highly expressed in these non-otic neural tissues, we detected no overt cellular abnormalities or degeneration in rda/rda mutant brains examined at 10 months of age. Electroretinography (ERG) tests of both rda/rda (tested at 3 months of age) and rda2J/rda2J (tested at 4 months of age) mutant mice showed normal retinal function. Polyclonal antibodies to a recombinant ELMOD1 protein detected an ELMOD1-specific band in western blots; however, it also detected additional non-specific bands (Figure 5D). Because of this cross-reactivity, the antibodies could not be used to localize sub-cellular ELMOD1-specific protein expression in tissue sec- tions. Discussion Elmod1 mutations and hair bundle abnormalities We used positional cloning to identify two independent, naturally occurring mutations of Elmod1, designated rda and rda2J. Both are complex DNA alterations; rda is a large 138 kb deletion with a small 756 bp inverted insertion at the point of the deletion, and rda2J is an in-frame intragenic duplication of exons 3–8. The rda deletion includes exons 1–5 of Elmod1, and as expected no Elmod1 gene products (either mRNA or protein) were detected in rda/rda mutant mice. Although wildtype ELMOD1 protein is absent in rda2J/rda2J mice, a larger mutant form of the ELMOD1 protein is produced; however, the extensive duplication of 202 amino acids in the mutant protein likely makes it nonfunctional. The similarity of the rda2J/rda2J mutant phenotype to that of rda/ rda mice provides additional evidence that rda2J is a allele. That two independent, complex DNA alterations like rda and rda2J occur at the same gene locus seems unusual, but there is some evidence that structural variants have a nonrandom genomic distribution, and that multiple independent mutations may arise in close proximity, perhaps at unstable genomic regions [24]. We found that Elmod1 expression localized to both cochlear and vestibular hair cells of the inner ear (Fig. 6), consistent with hair cell dysfunction being the underlying cause for the deafness and balance dysfunction of Elmod1 mutant mice. In support of our Elmod1 localization results, a recent study of cell type-specific transcriptomes from inner ear tissues of newborn mice identified Elmod1 as one of a large cluster of genes that is differentially expressed in auditory and vestibular sensory epithelia [25]. Elmod1 is expressed in vestibular hair cells of the crista ampullaris (Fig. 6B), which sense rotational movements of the head, thus their PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e36074