Meredith E. Adams

Loss of Chd7 function in gene-trapped reporter mice is embryonic lethal and associated with severe defects in multiple developing tissues.

CHD7 is a novel chromodomain gene mutated in 60%–80% of humans with CHARGE syndrome, a multiple congenital anomaly condition characterized by ocular coloboma, heart defects, atresia of the choanae, retarded growth and development, genital hypoplasia, and characteristic ear abnormalities including deafness. Phenotypic features of CHARGE are highly variable and incompletely penetrant. To explore developmental roles of CHD7, we generated mice carrying the Chd7Gt allele from a Chd7-deficient, gene-trapped lacZ reporter ES cell line. RT-PCR of embryo RNA demonstrated significantly reduced levels of wild-type transcript in Chd7Gt/Gt embryos. Chd7Gt/Gt embryos survive only up to embryonic day 10.5 (E10.5). Chd7Gt/+ male and female mice are viable, small, and exhibit variable degrees of head-bobbing and circling, consistent with vestibular dysfunction. Paint-filling of E16.5 heterozygous inner ears revealed defects of the semicircular canals. The pattern of β-galactosidase activity in Chd7Gt/+ embryos mimics Chd7 mRNA expression in wild-type embryos, confirming the fidelity of the lacZ reporter. We observed tissue-specific β-galactosidase in the E12.5 and E14.5 Chd7Gt/+ brain, pituitary, ear, heart, and craniofacial structures, indicating survival of Chd7Gt/+ cells in CHARGE-relevant organs. These studies demonstrate the utility of Chd7Gt as a reporter-tagged loss-of-function allele for future studies exploring developmental mechanisms of Chd7 deficiency.

Defects in Vestibular Sensory Epithelia and Innervation in Mice with Loss of Chd7 Function: Implications for Human CHARGE Syndrome

CHD7 is a chromodomain gene mutated in CHARGE syndrome, a multiple anomaly condition characterized by ocular coloboma, heart defects, atresia of the choanae, retarded growth and development, genital hypoplasia, and ear defects including deafness and semicircular canal dysgenesis. Mice with heterozygous Chd7 deficiency have circling behavior and semicircular canal defects and are an excellent animal model for exploring the pathogenesis of CHARGE features. Inner ear vestibular defects have been characterized in heterozygous Chd7‐deficient embryos and early postnatal mice, but it is not known whether vestibular defects persist throughout adulthood in Chd7‐deficient mice or whether the vestibular sensory epithelia and their associated innervation and function are intact. Here we describe a detailed analysis of inner ear vestibular structures in mature mice that are heterozygous for a Chd7‐deficient, gene‐trapped allele (Chd7Gt/+). Chd7Gt/+ mice display variable asymmetric lateral and posterior semicircular canal malformations, as well as defects in vestibular sensory epithelial innervation despite the presence of intact hair cells in the target organs. These observations have important functional implications for understanding the clinical manifestations of CHD7 mutations in humans and for designing therapies to treat inner ear vestibular dysfunction. J. Comp. Neurol. 504:519–532, 2007.

Reversible electrocochleographic abnormalities in superior canal dehiscence.

Objective: Electrocochleography (ECoG) is often used in the evaluation of episodic vertigo, and abnormal findings are commonly thought to be due to endolymphatic hydrops. We have observed that a number of patients with abnormal ECoG were ultimately found to have superior semicircular canal dehiscence (SSCD). Therefore, we examined the electrocochleographic findings in a series of patients with documented SSCD. Study Design: Retrospective case series. Setting: Academic medical center. Patients: Seven adult patients with unilateral SSCD and 4 patients with bilateral SSCD who underwent tympanic ECoG as part of a diagnostic or preoperative evaluation that also included vestibular-evoked myogenic potentials (VEMPs). Interventions: Patients underwent audiometric testing, ECoG, VEMP, and high-resolution temporal bone computed tomography reformatted to optimally view the superior semicircular canal. Five patients underwent superior canal obliteration. Postoperative VEMP and ECoG were performed in 4 of these patients. Intraoperative continuous ECoG was performed in 1 patient. Main Outcome Measures: Summating potential to action potential (SP/AP) ratio on ECoG. Results: Fourteen of 15 ears confirmed to have SSCD on computed tomographic imaging were found to have an elevated SP/AP ratio (defined as >0.40). In one patient with bilateral SSCD, the ear with the radiographically less severe dehiscence had an SP/AP ratio of 0.40, at the upper limit of normal, and a normal VEMP threshold. In all 4 patients who underwent obliteration of the dehiscent canal, and for whom postoperative test results were available, the SP/AP ratio normalized in the operated ear. In the 1 patient who underwent intraoperative ECoG, the SP/AP ratio normalized immediately after canal occlusion. Conclusion: An elevated SP/AP ratio seems to be a consistent finding in SSCD syndrome and, like the other abnormal audiometric and electrophysiologic findings associated with the syndrome, normalizes after surgical correction. Elevation of the SP/AP ratio has historically been associated with endolymphatic hydrops. The present findings expand the differential diagnosis of an abnormal ECoG and may shed light on the origin of an elevated summating potential.

Expression of p53 and Bcl-xL as predictive markers for larynx preservation in advanced laryngeal cancer

OBJECTIVE To assess tumor markers in advanced laryngeal cancer. DESIGN Marker expression and clinical outcome. PATIENTS Pretreatment tumor biopsy specimens were analyzed from patients enrolled in the Department of Veterans Affairs Laryngeal Cancer Study. MAIN OUTCOME MEASURES Expression of p53 (OMIM TP53) and Bcl-xL (OMIM 600039) in pretreatment biopsy specimens was assessed for correlation with chemotherapy response, laryngeal preservation, and survival. RESULTS Higher rates of larynx preservation were observed in patients whose tumors expressed p53 vs those that did not (80% [36 of 45 patients] vs 59% [24 of 41 patients], P =.03). Higher rates of larynx preservation were also observed in patients whose tumors expressed low levels of Bcl-xL vs high levels of Bcl-xL (90% [18 of 20 patients] vs 60% [30 of 50 patients], P =.02). Patients were categorized into 3 risk groups (low, intermediate, and high) based on their tumor p53 and Bcl-xL expression status. Patients whose tumors had the high-risk biomarker profile (low p53 expression and high Bcl-xL expression) were less likely to preserve their larynx than patients whose tumors had the intermediate-risk biomarker profile (high p53 expression and low or high Bcl-xL expression) or the low-risk biomarker profile (low p53 expression and low Bcl-xL expression). The larynx preservation rates were 100% (10 of 10 patients), 77% (26 of 34 patients), and 54% (7 of 13 patients) for the low-risk, intermediate-risk, and high-risk groups, respectively (P =.04, Fisher exact test). CONCLUSION Tumor expression of p53 and Bcl-xL is a strong predictor of successful larynx preservation in patients treated with induction chemotherapy and followed by radiation therapy in responding tumors.

Overexpression of cyclin D1 correlates with sensitivity to cisplatin in squamous cell carcinoma cell lines of the head and neck

Objective Treatment strategies for squamous cell carcinoma of the head and neck (SCCHN) are based on the TNM classification. Biological markers that can predict the response to therapy have so far not been introduced. The objective of this study was to investigate cyclin D1 deregulation relative to sensitivity to cisplatin. Material and methods This was a laboratory study of 23 established University of Michigan SCC cell lines. Chemosensitivity was assessed by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cyclin D1 amplification status was evaluated by real-time polymerase chain reaction (PCR; data were verified by differential and conventional PCR) using a chromosome 18q microsatellite marker probe (D18S70) as an internal control. Cyclin D1 protein expression was tested using Western blotting. Results Cyclin D1 amplification was seen in 9/23 (39%) and cyclin D1 overexpression in 12/19 (63%) of the cell lines. As expected, all cell lines showing amplification also showed overexpression of cyclin D1 (p=0.004; Fishers exact test). The mean cisplatin concentration inhibiting growth of 50% of the cells (ID50) was 9.8 μM in all cell lines (range 2.7–36.7 μM). Five of nine cell lines showing cyclin D1 amplification were highly sensitive to cisplatin (ID50 3–4.8 μM) and the remaining four revealed intermediate sensitivity. Five cell lines that strongly overexpressed cyclin D1 protein responded better to cisplatin than cell lines that showed any other expression (ID50 5.1 vs 11.2 μM; p=0.025; Students t-test). Conclusions This in vitro study suggests that overexpression of cyclin D1 is associated with a good response to cisplatin in SCC cell lines. Our results support the hypothesis that overexpression of cyclin D1 is one of the molecular factors that can be used to predict sensitivity to chemotherapy, thus enabling individualization of treatment of head and neck cancer.

Mature middle and inner ears express Chd7 and exhibit distinctive pathologies in a mouse model of CHARGE syndrome

Heterozygous mutations in the gene encoding chromodomain-DNA-binding-protein 7 (CHD7) cause CHARGE syndrome, a multiple anomaly condition which includes vestibular dysfunction and hearing loss. Mice with heterozygous Chd7 mutations exhibit semicircular canal dysgenesis and abnormal inner ear neurogenesis, and are an excellent model of CHARGE syndrome. Here we characterized Chd7 expression in mature middle and inner ears, analyzed morphological features of mutant ears and tested whether Chd7 mutant mice have altered responses to noise exposure and correlated those responses to inner and middle ear structure. We found that Chd7 is highly expressed in mature inner and outer hair cells, spiral ganglion neurons, vestibular sensory epithelia and middle ear ossicles. There were no obvious defects in individual hair cell morphology by prestin immunostaining or scanning electron microscopy, and cochlear innervation appeared normal in Chd7(Gt)(/+) mice. Hearing thresholds by auditory brainstem response (ABR) testing were elevated at 4 and 16 kHz in Chd7(Gt)(/+) mice, and there were reduced distortion product otoacoustic emissions (DPOAE). Exposure of Chd7(Gt)(/+) mice to broadband noise resulted in variable degrees of hair cell loss which inversely correlated with severity of stapedial defects. The degrees of hair cell loss and threshold shifts after noise exposure were more severe in wild type mice than in mutants. Together, these data indicate that Chd7(Gt)(/+) mice have combined conductive and sensorineural hearing loss, correlating with changes in both middle and inner ears.

Electrocochleography as a diagnostic and intraoperative adjunct in superior semicircular canal dehiscence syndrome

Objective To determine the electrocochleographic characteristics of ears with superior semicircular canal dehiscence (SSCD) and to examine its use for intraoperative monitoring in canal occlusion procedures. Study Design Case series. Setting Academic medical center. Patients Thirty-three patients (45 ears) had clinical and computed tomographic evidence of SSCD; 8 patients underwent intraoperative electrocochleography (ECoG) during superior canal occlusion; 9 patients underwent postoperative ECoG after SSCD occlusion. Interventions Diagnostic, intraoperative, and postoperative extratympanic ECoG; middle fossa or transmastoid occlusion of the superior semicircular canal. Main Outcome Measure Summating potential (SP) to action potential (AP) ratio, as measured by ECoG, and alterations in SP/AP during canal exposure and occlusion. Results Using computed tomography as the standard, elevation of SP/AP on ECoG demonstrated 89% sensitivity and 70% specificity for SSCD. The mean SP/AP ratio among ears with SSCD was significantly higher than that among unaffected ears (0.62 versus 0.29, p < 0.0001). During occlusion procedures, SP/AP increased on exposure of the canal lumen (mean change ± standard deviation, 0.48 ± 0.30). After occlusion, SP/AP dropped below the intraoperative baseline in most cases (mean change, −0.23 ± 0.52). All patients experienced symptomatic improvement. All patients who underwent postoperative ECoG 1 to 3 months after SSCD repair maintained SP/AP of 0.4 or lesser. Conclusion These findings expand the differential diagnosis of abnormal ECoG. In conjunction with clinical findings, ECoG may support a clinical diagnosis of SSCD. Intraoperative ECoG facilitates dehiscence documentation and allows the surgeon to confirm satisfactory canal occlusion.

Differences in the Diameter of Facial Nerve and Facial Canal in Bell's Palsy—A 3-Dimensional Temporal Bone Study

&NA; Bell’s palsy is hypothesized to result from virally mediated neural edema. Ischemia occurs as the nerve swells in its bony canal, blocking neural blood supply. Because viral infection is relatively common and Bell’s palsy relatively uncommon, it is reasonable to hypothesize that there are anatomic differences in facial canal (FC) that predispose the development of paralysis. Measurements of facial nerve (FN) and FC as it follows its tortuous course through the temporal bone are difficult without a 3D view. In this study, 3D reconstruction was used to compare temporal bones of patients with and without history of Bell’s palsy. Methods Twenty-two temporal bones (HTBs) were included in the study, 12 HTBs from patients with history of Bell’s palsy and 10 healthy controls. Three-dimensional models were generated from HTB histopathologic slides with reconstruction software (Amira), diameters of the FC and FN were measured at the midpoint of each segment. Results The mean diameter of the FC and FN was significantly smaller in the tympanic and mastoid segments (p = 0.01) in the BP group than in the controls. The FN to FC diameter ratio (FN/FC) was significantly bigger in the mastoid segment of BP group, when compared with the controls. When comparing the BP and control groups, the narrowest part of FC was the labyrinthine segment in control group and the tympanic segment in the BP. Conclusion This study suggests an anatomic difference in the diameter of FC in the tympanic and mastoid segments but not in the labyrinthine segment in patients with Bell’s palsy.

Capillary hemangioma of the tympanic membrane.

Capillary hemangiomas of the tympanic membrane are extremely rare benign vascular tumors. These lesions may be asymptomatic or associated with tinnitus, otalgia, or hearing loss. We present a case of an asymptomatic capillary hemangioma of the tympanic membrane excised without complications. An internal review board exemption was obtained. A 59-year-old woman with a history of bilateral nonpulsatile tinnitus and mild high-frequency sensorineural hearing loss presented to her local otolaryngologist for cleaning of recurrent cerumen impactions. She was found to have a vascular lesion on her left tympanic membrane that was not evident at her last cleaning. She noted no change in her hearing or tinnitus and denied trauma, aural fullness, otalgia, otorrhea, vertigo, and headache. She has no hemangiomas elsewhere. On physical examination, there was a 3-mm ovoid nonpulsatile lesion on the lateral surface of the tympanic membrane, just superior to the umbo (Fig. 1). The rest of the patients head and neck examination was unremarkable. Audiogram demonstrated bilateral symmetric mild high-frequency sensorineural hearing loss. Magnetic resonance imaging and computed tomography scans demonstrated a small lesion lateral to the tympanic membrane without extension into the middle ear. The patient was taken to the operating room for excisional biopsy. The lesion was removed, leaving the fibrous and mucosal layers intact. Pathologic examination showed a well-circumscribed lesion with small vascular spaces and endothelial cells within a fibrous stroma. The lesion was encapsulated on one side

Geographic Variation in Use of Vestibular Testing among Medicare Beneficiaries

Objective There is a lack of consensus regarding the indications for vestibular testing in the evaluation of dizziness and balance disorders. Geographic variation in health services utilization is associated with lack of consensus. To understand the variation in current practice, we investigated the patterns of use of vestibular testing and diagnosis codes for dizziness and balance disorders among individuals ≥65 years of age across different regions of the United States. Study Design Cross-sectional study. Setting Medicare administrative claims data. Subjects and Methods Using the Summarized Denominator file, a sample of the US population linked to the Surveillance, Epidemiology, and End Results (SEER)–Medicare files (years 2000-2010), we identified persons who were ≥65 years of age. We used multivariable analyses to determine the factors associated with vestibular testing and diagnoses. Results Of the 231,984 eligible Medicare beneficiaries, 27% were diagnosed with dizziness and balance disorders. Patterns of use of vestibular tests (eye movement recording for spontaneous nystagmus, caloric testing, and rotary chair testing) varied significantly by geographic region. Rotary chair test utilization varied most. We found significant geographic variation in vestibular testing and diagnoses after controlling for age, sex, race, Medicaid participation, and rurality. Conclusions There may be opportunities to improve the consistency and efficiency of care for dizziness and balance disorders. It will be important to define appropriate levels of vestibular diagnostic testing and which tests add sufficient value to justify the costs. Further work is needed to better characterize the causes and consequences of variation in vestibular test utilization.