The first USH2A mutation analysis of Japanese autosomal recessive retinitis pigmentosa patients: a totally different mutation profile with the lack of frequent mutations found in Caucasian patients.

Retinitis pigmentosa (RP) is a highly heterogeneous genetic disease. The USH2A gene, which accounts for approximately 74-90% of Usher syndrome type 2 (USH2) cases, is also one of the major autosomal recessive RP (arRP) causative genes among Caucasian populations. To identify disease-causing USH2A gene mutations in Japanese RP patients, all 73 exons were screened for mutations by direct sequencing. In total, 100 unrelated Japanese RP patients with no systemic manifestations were identified, excluding families with obvious autosomal dominant inheritance. Of these 100 patients, 82 were included in this present study after 18 RP patients with very likely pathogenic EYS (eyes shut homolog) mutations were excluded. The mutation analysis of the USH2A revealed five very likely pathogenic mutations in four patients. A patient had only one very likely pathogenic mutation and the others had two of them. Caucasian frequent mutations p.C759F in arRP and p.E767fs in USH2 were not found. All the four patients exhibited typical clinical features of RP. The observed prevalence of USH2A gene mutations was approximately 4% among Japanese arRP patients, and the profile of the USH2A gene mutations differed largely between Japanese patients and previously reported Caucasian populations.

A manual of Eustachian tube function tests-illustration of representative test results obtained from healthy subjects and typical disorders with suggestion of the appropriate test method of choice.

In the 19th century, Politzer devised a method to measure passage of the Eustachian tube (ET) by pressurizing the nasopharyngeal cavity, which marked the beginning of the ET function test. Since then, various examination methods have been developed. While ET function testing is important, recent advancements in diagnostic imaging and treatments have renewed interest on its importance. In Japan, the main objective methods used for examining ET function include tubotympanoaero-dynamic graphy (TTAG), sonotubometry, and the inflation-deflation test. The Japan Otological Society (JOS) Eustachian Tube Committee has proposed a manual of ET function tests, which presents typical patterns of the normal ear and typical diseases and suggests the ET function test of choice for each disease. However, the diagnosis of each disease should be made based on a comprehensive history and various examination findings, with ET function tests playing a supplemental role in the diagnosis.

Congenital middle ear cholesteatoma: experience from 26 surgical cases.

OBJECTIVES: We analyzed the clinical features and surgical techniques used in cases of childhood congenital cholesteatoma of the middle ear. METHODS: We studied 26 patients (26 ears) who underwent surgery for congenital cholesteatoma between January 1998 and December 2009, focusing on the location and type of cholesteatoma, the surgical procedures involved, and the results obtained. Patients with prior otologic procedures were excluded. A 4-stage system was used to grade the cholesteatomas. RESULTS: The frequency of posterior-quadrant involvement and open-type cholesteatomas increased in the more advanced stages. Second-look operations were performed in 60% of stage III and 75% of stage IV cases; and residual cholesteatomas were found in 20% of stage III and 75% of stage IV cases. Of the cases evaluated both before and after the operation, 100% of stage I and II cases, 86% of stage III cases, and 50% of stage IV cases showed improvement in hearing function. CONCLUSIONS: The staging system is relatively simple, while accurately reflecting clinical results. However, there are many differences between the anterior and posterior types of congenital cholesteatomas in surgical approach and postoperative progression that are not reflected in the classification systems and require further study. In addition, we reviewed the surgical procedures involved in anterior-quadrant cases, and propose a modified surgical procedure.

Novel USH2A mutations in Japanese Usher syndrome type 2 patients: marked differences in the mutation spectrum between the Japanese and other populations.

Usher syndrome (USH) is an autosomal recessive disorder characterized by retinitis pigmentosa and hearing loss. USH type 2 (USH2) is the most common type of USH and is frequently caused by mutations in USH2A. In a recent mutation screening of USH2A in Japanese USH2 patients, we identified 11 novel mutations in 10 patients and found the possible frequent mutation c.8559-2A>G in 4 of 10 patients. To obtain a more precise mutation spectrum, we analyzed further nine Japanese patients in this study. We identified nine mutations, of which eight were novel. This result indicates that the mutation spectrum for USH2A among Japanese patients largely differs from Caucasian, Jewish and Palestinian patients. Meanwhile, we did not find the c.8559-2A>G in this study. Haplotype analysis of the c.8559-2G (mutated) alleles using 23 single nucleotide polymorphisms surrounding the mutation revealed an identical haplotype pattern of at least 635 kb in length, strongly suggesting that the mutation originated from a common ancestor. The fact that all patients carrying c.8559-2A>G came from western Japan suggests that the mutation is mainly distributed in that area; indeed, most of the patients involved in this study came from eastern Japan, which contributed to the absence of c.8559-2A>G.

Specific localization of five phosphatidylcholine species in the cochlea by mass microscopy.

Phosphatidylcholine (PC), a phospholipid, is a basic structural component of cell membranes. PC species exhibit various binding patterns with fatty acids; however, the distributions of PC species have not been studied in the cochlea. In recent years, imaging mass spectrometry has been used as a biomolecular visualization technique in medical and biological sciences. We recently developed a 'mass microscope' consisting of a mass spectrometry imager with high spatial resolution equipped with an atmospheric-pressure matrix-assisted laser desorption/ionization and quadrupole ion trap time-of-flight analyzer. In this study, we applied the mass microscope to analyze cochlear tissue sections. The imager allowed visualization of the localization of PC species in each region of the cochlea. The structures of the PC species were determined using tandem mass spectrometry. PC(16:0/18:1) was highly localized in the organ of Corti and the stria vascularis. PC(16:0/18:2) was mainly observed in the spiral ligament. PC(16:0/16:1) was found primarily in the organ of Corti. These distributional differences may be associated with the cellular architecture of these cochlear regions.

Hair roots as an mRNA source for mutation analysis of Usher syndrome-causing genes.

mRNA is an important tool to study the effects of particular mutations on the mode of splicing and transcripts. However, it is often difficult to isolate mRNA because the organ or tissue in which the gene is expressed cannot be sampled. We previously identified two probable splicing mutations (c.6485+5G>A and c.8559-2A>G) during the mutation analysis of USH2A in Japanese Usher syndrome (USH) type 2 patients, but we could not observe their effects on splicing because the gene is expressed in only a few tissues/organs, and is not expressed in peripheral lymphocytes. In this study, we used hair roots as a source of mRNA of USH-causing genes, and successfully detected the expression of seven, except USH1C and CLRN1, of the nine USH-causing genes. We used RNA extracted from the hair roots of a patient who has both c.6485+5G>A and c.8559-2A>G mutations in USH2A in a compound heterozygous state to observe the effects of these mutations on transcripts. Reverse-transcription PCR analysis revealed that c.6485+5G>A and c.8559-2A>G inactivated splice donor and splice acceptor sites, respectively, and caused skipping of exons. Thus, RNA extracted from hair roots is a potential powerful and convenient tool for the mutation analysis of USH-causing genes.

Mutation analysis of the MYO7A and CDH23 genes in Japanese patients with Usher syndrome type 1.

Usher syndrome (USH) is an autosomal recessive disorder characterized by retinitis pigmentosa and hearing loss. USH type 1 (USH1), the second common type of USH, is frequently caused by MYO7A and CDH23 mutations, accounting for 70-80% of the cases among various ethnicities, including Caucasians, Africans and Asians. However, there have been no reports of mutation analysis for any responsible genes for USH1 in Japanese patients. This study describes the first mutation analysis of MYO7A and CDH23 in Japanese USH1 patients. Five mutations (three in MYO7A and two in CDH23) were identified in four of five unrelated patients. Of these mutations, two were novel. One of them, p.Tyr1942SerfsX23 in CDH23, was a large deletion causing the loss of 3 exons. This is the first large deletion to be found in CDH23. The incidence of the MYO7A and CDH23 mutations in the study population was 80%, which is consistent with previous findings. Therefore, mutation screening for these genes is expected to be a highly sensitive method for diagnosing USH1 among the Japanese.

Ultrastructural localization of cochlin in the rat cochlear duct.

Cochlin, a product of the COCH gene, is a major constituent of the inner ear extracellular matrix. Type II collagen, a protein that contributes to structural stability, is also a component of this extracellular matrix. In this study, using the postembedding immunogold method, we demonstrate the localization of cochlin and type II collagen in the cochlear duct at the ultrastructural level. The immunolabeling of cochlin was observed in the fibrillar substance in the spiral limbus, beneath the inner sulcus cells, and in the basilar membrane, the spiral prominence and the spiral ligament. Immunolabeling of type II collagen was observed in the same fibrillar substance in the extracellular matrix of the cochlear duct. This localization of cochlin is consistent with the expected localization of type II collagen. The localization of cochlin and type II collagen indicates the important roles played by these proteins in the hearing process.

Postoperative Course of Pars Flaccida Cholesteatoma Patients With Habitual Sniffing.

OBJECTIVE: To investigate the effects of habitual sniffing on the postoperative course of pars flaccida cholesteatoma. STUDY DESIGN: Retrospective case series study. SETTING: University hospital. PATIENTS: Forty-nine patients (53 ears) with pars flaccida cholesteatoma and history of habitual sniffing before the initial operation. INTERVENTIONS: Patients were divided into a "sniffing cessation group" characterized by sniffing cessation and a "continual sniffing group" characterized by continuation of sniffing despite instructions for conscious cessation. MAIN OUTCOME MEASURES: Hearing level, tympanic membrane findings, tympanograms, mastoid cell development before the operation, and pneumatization 1 year postoperatively. RESULTS: The sniffing cessation and continual sniffing groups comprised 35 patients (38 ears) and 14 patients (15 ears), respectively. The average postoperative hearing was slightly better in the continual sniffing group. In the sniffing cessation group, retractions were evident in significantly fewer cases. Retractions were observed in all continual sniffing group cases, with a high percentage of severe retractions, wherein the bottom was not visible. Type A tympanogram was predominant in the sniffing cessation group. Mastoid cell development was not significantly different between the two groups. Satisfactory pneumatization was significantly more common in the sniffing cessation group (Fisher's exact test, p < 0.005). CONCLUSION: Conscious cessation of the sniffing habit could reduce the risk of postoperative retraction and improve pneumatization in patients with pars flaccida cholesteatoma. The presence or absence of the sniffing habit after surgery is a defining factor in postoperative prognosis (retraction, recurrence), and may be a determinant for decisions regarding surgical approach.

Efficacy of a silicone plug for patulous eustachian tube: A prospective, multicenter case series.

OBJECTIVES/HYPOTHESIS: To report the efficacy and safety of transtympanic plugging of the eustachian tube (ET) using a silicone plug (Kobayashi plug) for chronic patulous ET (PET). STUDY DESIGN: Prospective and multicenter trial conducted in which 30 PET patients were resistant to at least 6 months of conservative treatment. METHODS: The efficacy and safety of 28 and 27 patients, respectively, were analyzed. All patients fulfilled inclusion and exclusion criteria. The primary end point used the patulous eustachian tube handicap inventory-10 (PHI-10), and the secondary end point used ET function tests such as sonotubometry, tubo-tympano-aerodynamic-graphy, and respiratory movement of the tympanic membrane and auscultation of voice sounds transmitted from the nose through the ET to the external auditory canal at 3months after surgery. RESULTS: PHI-10 scores were 34.4 ± 4.2, 6.4 ± 9, and 5.7 ± 8.6 at screening, and 3 and 6 months after surgery. Twenty-three cases (82.1%, 95% confidence interval: 63.1%-93.9%) were judged as successes. There were five cases (17.2%) of middle ear effusion, four cases (13.8%) of tympanic membrane perforation, and one case of tinnitus due to surgery to remove the plug. No severe or life-threatening complications were found. CONCLUSIONS: This study revealed the efficacy and safety of silicone plug insertion for severe PET patients. LEVEL OF EVIDENCE: 2 Laryngoscope, 130:1304-1309, 2020.

Cochlear implant in children with asymptomatic congenital cytomegalovirus infection.

OBJECTIVE: To evaluate the development of speech perception and auditory skills after cochlear implantation in deaf children with asymptomatic congenital cytomegalovirus (CMV) infection diagnosed based on the presence of CMV DNA in the neonatal urine. STUDY DESIGN: A prospective study of congenital CMV infection was done between 1996 and 2003. Of 18 children diagnosed with congenital CMV infection, 2 deaf children with asymptomatic CMV infections received cochlear implantation. RESULTS: The 2 deaf children who received cochlear implantation had delayed-onset, progressive sensorineural hearing loss on follow-up audiometric examinations administered at 29 and 39 months of age. After cochlear implantation, their Infant-Toddler Meaningful Auditory Integration Scale scores increased consistently during 36 months of follow-up; these results were similar to those of 5 congenitally deaf children without CMV infection who had cochlear implantation. CONCLUSIONS: Cochlear implantation was effective for improving the development of speech perception and auditory skills in deaf children with asymptomatic congenital CMV infection. There was no significant difference in the development of useful auditory integration between our general pediatric cochlear implant population without CMV infection and those with asymptomatic CMV infection.

Spatiotemporal expression of cochlin in the inner ear of rats during postnatal development.

Cochlin (encoded by COCH) constitutes 70% of non-collagenous protein in the inner ear, and the expression of cochlin is highly specific to the inner ear. Eleven missense mutation and one in-frame deletion have been reported in the COCH gene, causing hereditary progressive sensorineural hearing loss and vestibular dysfunction, DFNA9. These data imply that cochlin should bear an essential and crucial role in the inner ear function. However, the role of cochlin has not been fully clarified. We have investigated the spatiotemporal expression of cochlin in the inner ear of rats during postnatal development to better understand the functional role of cochlin. By immunohistochemistry, cochlin expression was faint in the cochlea and vestibule on the 6th day after birth (DAB6). At DAB70, strong expression of cochlin was detected in the spiral limbus and spiral ligament within the cochlea, and in the stromata of the maculae of otolithic organs and crista ampullaris within the vestibule. Immunoreactivity for cochlin increased during the postnatal development. Western blot analysis also showed an increase in the expression of cochlin isoforms. Furthermore, the dominant isoform of cochlin expressed changed from p63s to p40s between DAB24 and DAB70. These results suggest that the expression of cochlin may be related to the maturation of inner ear function, and the change in isoforms of cochlin expressed will provide important insight into the understanding of both cochlin function and formation of cochlin isoforms. This is the first to report about the spatiotemporal expression of cochlin in the developing rat inner ear.

Ultrastructural co-localization of cochlin and type II collagen in the rat semicircular canal.

Cochlin and type II collagen are major constituents of the inner ear extracellular matrix. To investigate the morphological relation of cochlin and type II collagen in the rat semicircular canal, immuno-electronmicroscopic analysis was performed using the post-embedding immunogold method. Immunolabeling for cochlin was detected in the fibrillar substance underlying the supporting epithelium of the sensory cells and beneath the epithelial cells facing the endolymph in the semicircular canals. Immunolabeling for type II collagen was observed in the same fibrillar substance in the subepithelial area. The co-localization of cochlin and type II collagen in the fibrillar substance in the subepithelial area indicate that cochlin may play a role in the structural homeostasis of the vestibule acting in concert with the fibrillar type II collagen bundles.

Localization of megalin in rat vestibular dark cells and endolymphatic sac epithelial cells.

CONCLUSION: Megalin immunoreactivity was observed in kidney proximal tubule cells, vestibular dark cells, and epithelial cells of the endolymphatic sac. Endocytic mechanisms appear to differ between the endolymphatic sac and proximal tubule cells. We speculate that megalin is secreted by a certain type of cell into the endolymphatic space, and is then absorbed from the endolymphatic space by another type of cell to maintain endolymphatic sac homeostasis. OBJECTIVES: We previously detected megalin immunoreactivity in the rat cochlear duct. Megalin may be involved in endocytosis in the vestibular organ and endolymphatic sac. To examine this possibility, we extended our immunocytochemical investigation to the rat inner ear cells with special attention to vestibular dark cells and endolymphatic sac. MATERIALS AND METHODS: We observed immunoreactivity of megalin under light and electron microscopy. The primary antibody was rabbit polyclonal antibody that had been raised against rat immunoaffinity-purified megalin. RESULTS: The luminal membrane and subapical area of dark cells in the semicircular canal were immunolabeled. The stainable substance in the endolymphatic space was strongly stained. The cytoplasm of epithelial cells was also stained in various patterns.

A case of improved hearing with cochlear implantation in Gaucher disease type 1.

Gaucher disease is a lysosomal storage disorder that is caused by congenital defective function of the enzyme glucocerebrosidase. Glucocerebroside that is not hydrolyzed by glucocerebrosidase mainly accumulates in the reticular tissue. We describe a Japanese boy with Gaucher disease type 1 who developed bilateral profound sensorineural hearing loss within approximately 4years. We performed cochlear implantation initially on his right ear and again on his left ear 5 months later. The cochlear implants were successfully utilized with a speech discrimination score of 95% on a Japanese sentence recognition test. There are many reports of central hearing loss in Gaucher disease type 2 or 3. However, to the best of our knowledge, this is the first report of profound inner ear hearing loss with Gaucher disease. It also appears to be the first record of cochlear implantation for Gaucher disease. Cochlear implants may be useful for sensorineural hearing loss in patients with Gaucher disease without neurological symptoms other than hearing loss.

Visual Outcomes in Japanese Patients with Retinitis Pigmentosa and Usher Syndrome Caused by USH2A Mutations.

PURPOSE: EYS and USH2A are the most common causative genes for retinitis pigmentosa (RP) in Japan. We determined the clinical outcomes for USH2A-related non-syndromic RP or Usher syndrome type II (USH2). METHODS: Two non-syndromic RP and 11 USH2 patients with previously identified USH2A mutations were included. Their complete history and medical records were collected using standard procedures. Visual fields and acuity were compared with those of patients with EYS mutations. Clinical analyses were based on ophthalmic and otolaryngologic examinations. RESULTS: In all patients, the fundus displayed changes typical of RP. Most patients showed relatively well-preserved visual acuity in their thirties or forties, with rapid deterioration in their fifties. Concentric constriction started in the twenties or thirties, and no effective residual visual field was observed after the fifties. CONCLUSIONS: The visual outcome for non-syndromic RP or USH2 patients with USH2A mutations is consistent with that for RP patients with EYS mutations.

Diagnostic criteria for Patulous Eustachian Tube: A proposal by the Japan Otological Society.

Patulous Eustachian Tube (PET) is of increasing importance in otology. However, despite the abundance of diseases requiring a differential diagnosis from PET, such as superior semicircular canal dehiscence syndrome, perilymphatic fistula, acute low-tone sensorineural hearing loss, etc., there are currently no established diagnostic criteria for PET. In view of these circumstances, the Japan Otological Society (JOS) Eustachian Tube Committee proposed the diagnostic criteria for Patulous Eustachian Tube in 2012, in order to promote clinical research on PET. A revision was made in 2016, maintaining the original concept that the criteria should be very simple, avoid any contamination of "Definite PET" with uncertain cases. Moreover, it was also intended to minimize the number of cases that could be accidentally excluded even in the presence of some suspected findings ("Possible PET"). The criteria can be used by all otolaryngologists even without using the Eustachian tube function test apparatus. However, the use of such an apparatus may increase the chances of detecting "Definite PET". The algorithm for the diagnosis of PET using the criteria has also been described. The JOS diagnostic criteria for Patulous Eustachian Tube will further promote international scientific communication on PET.

Pattern of cochlear damage caused by short-term kanamycin application using the round window microcatheter method.

CONCLUSION: Short-term local application of kanamycin by the microcatheter method can control the region and severity of drug effect on the cochlea by changing the concentration and administration time. OBJECTIVES: To investigate the effect of short-term round window administration of kanamycin by the microcatheter method on cochlear stereociliary bundle loss in a guinea pig model. MATERIALS AND METHODS: Two concentrations and two time periods were used for drug administration to the inner ear. In groups of six animals, a total dose of 172.5 mg/ml or 345 mg/ml of kanamycin was instilled at the rate of 0.1 ml/h into the round window niche over either a 1 h or 2 h period by the microcatheter method. One group of six animals received a saline infusion as a control. The animals were sacrificed after 4 days and stereociliary bundle loss was observed by SEM. Functional changes were evaluated by auditory brainstem response. RESULTS: With regard to the severity of the damage, higher drug concentrations were associated with more severe effects. The severity of damage was the same when the same total dose was used; however, it was found that when the dose was administered over a longer period, the damage region was wider. The functional changes also corresponded with the morphological changes.

The effect of ventilation tube insertion or trans-tympanic silicone plug insertion on a patulous Eustachian tube.

Conclusions This study suggests that long-term ventilation tube insertion is the first-choice surgical treatment for a 'sniff-type' patulous Eustachian tube (PET). When treating a refractory PET, it is important to determine whether the patient had a habitual sniff. Objectives PET patients were divided into two groups: patients with a habitual sniff (sniff-type PET) and those without a habitual sniff (non-sniff-type PET). This study examined the effects of ventilation tube insertion or silicone plug insertion in each group. Methods Surgical procedures such as ventilation tube insertion or trans-tympanic silicone plug insertion were performed for these patients. Tubotympanoaero-dynamic graphy (TTAG) was also performed to determine the mechanisms underlying these treatments. Results There were 11 cases (17 ears) of sniff-type PET and 20 cases (27 ears) of non-sniff-type PET. An improvement in symptoms was found in 72.7% of the patients who underwent silicone plug insertion (66.7% for sniff-type PET and 74.1% for non-sniff-type PET) and in 90.9% of the patients who underwent ventilation tube insertion for sniff-type PET. In TTAG assessments, many sniff-type PET patients showed significant synchronous changes at high levels of pressure (over 40 daPa) in the external auditory meatus and nasopharynx when performing a slight Valsalva manoeuvre (below 200 daPa).