Background: Perilymphatic fistula (PLF) is an abnormal connection between the inner and middle ear. A procedure for obtaining definite proof of a PLF remains elusive, and methods of diagnosis remain controversial. To date, there is no clinically relevant biochemical marker for perilymph leakage. Using proteomic analysis of inner ear proteins, we have previously found unique properties of cochlin, encoded by the COCH gene. We detected 3 cochlin isoforms (p63s, p44s and p40s) in the inner ear tissue and a short 16-kDa isoform of cochlin-tomoprotein (CTP) in the perilymph. Since cochlin was found to be highly specific to the inner ear, we speculated that CTP might also be specific to the perilymph. The aim of this study was to determine whether CTP, a novel perilymph-specific protein, could be used as a marker for the diagnosis of PLF. Methods: By Western blotting, we investigated the specificity of CTP expression in a range of body fluids that included perilymph, serum, saliva and cerebrospinal fluid. To elucidate the detection limit of CTP, serially diluted recombinant human (rh)CTP as well as human perilymph was tested. Results: CTP was selectively expressed in all 20 perilymph samples tested, but not in 77 samples of the other body fluids. The detection limit of rhCTP was 0.27 ng or 0.022 μl of perilymph per well on Western blot analysis. Conclusion: The results strongly suggest that CTP can be a specific marker of perilymph leakage. Moreover, CTP has the potential to be a biochemical marker that allows a definitive diagnosis of the etiology of PLF-related hearing loss and vestibular disorders.

A total of seven individuals were included in this study. CTP was detected in three of four cases with posterosuperior quadrant perforation of the tympanic membrane. In one of these three cases, even though the high resolution CT scan was not suggestive of PLF and the perilymph leakage could not be visualized intraoperatively, the CTP detection test was able to detect PLF. In two cases, the preoperative positive test results enabled us to make a diagnosis of PLF and a decision for surgical treatment. CTP was not detected in the cases with anterior or inferior tympanic membrane perforation.

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Proteomic analysis of inner ear proteins revealed unique properties of cochlin, encoded by the COCH gene. We detected 3 cochlin isoforms, p63s, p44s and p40s, in the inner ear tissue and a short 16-kDa isoform, cochlin-tomoprotein (CTP), in the perilymph. The role of the cochlin isoforms has not been elucidated. To improve our understanding of the mechanism of cochlin isoform expression, we investigated rat cochlin mRNA expression in the inner ear and other organs. We performed RNA-ligation-mediated amplification of cDNA ends (RLM-RACE) using RNA isolated from the inner ear and spleen of rats, which are known to express abundant cochlin mRNA. We also examined the expression profile of full-length cochlin mRNA by nested RT-PCR in the cerebrum, cerebellum/brain stem, eye, inner ear, thyroid gland, thymus gland, lung, heart, liver, spleen, adrenal gland, kidney and blood. We verified CTP expression in rat perilymph by Western blot. By RLM-RACE, alternately spliced variants of cochlin mRNA with 3 different lengths were detected (2442, 2008 and 724 bp). The two longer mRNAs encode full-length cochlin with different polyadenylation signals in the 3′-untranslated region, which are expressed both in the ear and spleen. The short variant encodes the limulus factor C, cochlin, late gestation lung protein (LCCL) domain and the N-terminal sequence of the von Willebrand factor A (vWFA1) domain, and this variant was detected only in the ear. All 3 variants have the same transcriptional start site. By RT-PCR, we found that full-length cochlin was expressed in all organs examined, with a splice variant in the heart. By Western blot, we detected short isoforms (11–17 kDa) in the perilymph. Cochlin isoform formation is regulated, at least in part, by alternative splicing at the transcriptional level. The short mRNA was detected only in the inner ear, and this variant may provide a clue to understanding the formation and function of cochlin isoforms.

The molecular mechanisms underlying age-related hearing loss are unknown, and currently, there is no treatment for this condition. Recent studies have shown that microRNAs (miRNAs) and age-related diseases are intimately linked, suggesting that some miRNAs may present attractive therapeutic targets. In this study, we obtained 8 human temporal bones from 8 elderly subjects at brain autopsy in order to investigate the expression profile of miRNAs in the inner ear with miRNA arrays. A mean of 478 different miRNAs were expressed in the samples, of which 348 were commonly expressed in all 8 samples. Of these, levels of 16 miRNAs significantly differed between young elderly and old elderly subjects. miRNAs, which play important roles in inner ear development, were detected in all samples, i.e., in both young and old elderly subjects, whether with or without hearing loss. Our results suggest that these miRNAs play important roles not only in development, but also in the maintenance of inner ear homeostasis.