High-throughput detection of mutations responsible for childhood hearing loss using resequencing microarrays

Background: Despite current knowledge of mutations in 45 genes that can cause nonsyndromic sensorineural hearing loss (SNHL), no unified clinical test has been developed that can comprehensively detect mutations in multiple genes. We therefore designed Affymetrix resequencing microarrays capable of resequencing 13 genes mutated in SNHL (GJB2, GJB6, CDH23, KCNE1, KCNQ1, MYO7A, OTOF, PDS, MYO6, SLC26A5, TMIE, TMPRSS3, USH1C). We present results from hearing loss arrays developed in two different research facilities and highlight some of the approaches we adopted to enhance the applicability of resequencing arrays in a clinical setting. Results: We leveraged sequence and intensity pattern features responsible for diminished coverage and accuracy and developed a novel algorithm, sPROFILER, which resolved >80% of no-calls from GSEQ and allowed 99.6% (range: 99.2-99.8%) of sequence to be called, while maintaining overall accuracy at >99.8% based upon dideoxy sequencing comparison. Conclusions: Together, these findings provide insight into critical issues for disease-centered resequencing protocols suitable for clinical application and support the use of array-based resequencing technology as a valuable molecular diagnostic tool for pediatric SNHL and other genetic diseases with substantial genetic heterogeneity

A rapid method for detection of five known mutations associated with aminoglycoside-induced deafness

<p>Abstract</p> <p>Background</p> <p>South Africa has one of the highest incidences of multidrug-resistant tuberculosis (MDR-TB) in the world. Concomitantly, aminoglycosides are commonly used in this country as a treatment against MDR-TB. To date, at least five mutations are known to confer susceptibility to aminoglycoside-induced hearing loss. The aim of the present study was to develop a rapid screening method to determine whether these mutations are present in the South African population.</p> <p>Methods</p> <p>A multiplex method using the SNaPshot technique was used to screen for five mutations in the <it>MT-RNR1 </it>gene: A1555G, C1494T, T1095C, 961delT+C(n) and A827G. A total of 204 South African control samples, comprising 98 Mixed ancestry and 106 Black individuals were screened for the presence of the five mutations.</p> <p>Results</p> <p>A robust, cost-effective method was developed that detected the presence of all five sequence variants simultaneously. In this pilot study, the A1555G mutation was identified at a frequency of 0.9% in the Black control samples. The 961delT+C(n) variant was present in 6.6% of the Black controls and 2% of the Mixed ancestry controls. The T1095C, C1494T and A827G variants were not identified in any of the study participants.</p> <p>Conclusion</p> <p>The frequency of 0.9% for the A1555G mutation in the Black population in South Africa is of concern given the high incidence of MDR-TB in this particular ethnic group. Future larger studies are warranted to determine the true frequencies of the aminoglycoside deafness mutations in the general South African population. The high frequencies of the 961delT+C(n) variant observed in the controls suggest that this change is a common non-pathogenic polymorphism. This genetic method facilitates the identification of individuals at high risk of developing hearing loss prior to the start of aminoglycoside therapy. This is important in a low-resource country like South Africa where, despite their adverse side-effects, aminoglycosides will continue to be used routinely and are accompanied with very limited or no audiological monitoring.</p

Laryngomalacia and its treatment

Objective: To determine 1) airway outcome of infants with laryngomalacia who do not undergo routine direct laryngoscopy (DL) and bronchoscopy (B), 2) the age at resolution of laryngomalacia, and, 3) outcome of supraglottoplasty as a function of the type of laryngomalacia and the presence of concomitant disease. Study Design: Retrospective chart review. Methods: The records of all infants diagnosed with laryngomalacia by flexible fiberoptic laryngoscopy (FFL) between 1990 and 1998 in the Department of Otolaryngology - Head and Neck Surgery, University of Iowa (Iowa City, IA) were reviewed. The type of laryngomalacia was designated by a new classification scheme (types 1-3) based on the site of supraglottic obstruction and the type of supraglottoplasty indicated, should the patient later require surgical intervention. The log rank test was used to compare age at resolution and outcome between types of laryngomalacia and between infants with isolated laryngomalacia versus those with additional congenital abnormalities and/or severe neurological compromise. Results: The type of laryngomalacia was evident in 48 of the 58 charts reviewed and included type 1 (57%), type 2 (15%), type 3 (13%), or combined types (15%). Twenty percent had severe neurological compromise and/or multiple congenital anomalies. The median time to resolution of stridor in these patients was not significantly delayed when compared with infants who had isolated airway anomalies (36 and 72 wk, respectively, vs. 36 wk for isolated laryngomalacia; P \u3c .4). Time to resolution did not correlate with the type of laryngomalacia. In 22 infants, clinical symptoms or findings suggested a synchronous airway lesion, and direct laryngoscopy and bronchoscopy were performed. In 11 infants, a second airway lesion was diagnosed (in four cases by FFL and in 7 cases by direct laryngoscopy and bronchoscopy). Complications did not arise in infants who did not undergo direct laryngoscopy and bronchoscopy. Eleven infants with severe laryngomalacia required surgical intervention. The success of supraglottoplasty did not correlate with the type of laryngomalacia or the presence of other congenital anomalies. Conclusions: Routine direct laryngoscopy and bronchoscopy as part of the evaluation of laryngomalacia are not warranted. Performing these procedures should be based on clinical and physical evidence of a concomitant airway lesion. In general, laryngomalacia will resolve within the first year of life, even in children with multiple congenital anomalies and/or severe neurological compromise. The proposed classification scheme is advantagous in that it is simple and correlates the site of obstruction with the surgical procedure most likely to effect a cure, should the patient require a supraglottoplasty. Surgical management is necessary in approximately 15% to 20% of affected infants

An update on the treatment of hemangiomas in children with interferon alfa-2a

Objective: To report the benefits and complications of subcutaneous interferon alfa-2a therapy for hemangiomas in children. Design: Prospective nonrandomized trial. Setting: Tertiary care pediatric referral center. Patients: Twenty-four pediatric patients diagnosed with massive or life- threatening hemangiomas. Interventions: Each patient received daily subcutaneous injections of interferon alfa-2a to a target close of 3 million U/m2 of booty surface area for a minimum of 4 months. Nineteen patients completed therapy and have received adequate follow-up. Main Outcome Measures: Clinical and radiographic comparisons before, during, and after therapy. Reduction in hemangioma size was graded as complete (\u3e90%), substantial (50%-80%), intermediate (20%-40%), or no response (\u3c 10%). Results: Mean age at institution of therapy was 9.6 months, and mean duration of treatment was 10.2 months. Most patients (70%) had not received prior therapy. Responses were as follows: complete, 8 patients (42%); substantial, 3 patients (16%); intermediate, 5 patients (26%); and no response, 3 patients (16%) (n = 19). During therapy, 5 patients (26%) developed neurological abnormalities: 3 had an unsteady gait, and 2 had fine motor deficits. Only 1 of these 5 patients required premature termination from the study, and the neurological abnormalities in all 5 patients resolved after treatment was discontinued. Two of the 4 patients with neurological findings who completed therapy demonstrated complete resolution of their hemangiomas. Patients who developed neurological abnormalities began interferon alfa-2a therapy at an earlier age (4.7 months) than patients without neurological difficulties (aged 11.1 months). The mean time from initiation of therapy to the appearance of neurological complications was 4.8 months. Conclusions: In pediatric patients with massive or life-threatening hemangiomas, interferon alfa-2a therapy is an effective treatment option. However, neurological evaluation before and during therapy with interferon alfa-2a should be performed owing to a significant incidence of neurological abnormalities (28%). Although all children with neurological findings demonstrated neurological recovers after discontinuation of therapy, we have changed our protocol and now more gradually increase the dosage of interferon alfa-2a up to 3 million U/m2 per day. The effect of this modification on the development of neurological abnormalities has not yet been determined

Nontraumatic atlantoaxial rotary subluxation in the pediatric otolaryngology patient: A report of four cases

Nontraumatic atlantoaxial rotary subluxation (NAARS) is a relatively uncommon entity, with inconsistent presentations. It most commonly follows infectious processes or operative procedures. We present our experience with 4 pediatric otolaryngology patients with NAARS who were treated at the University of Iowa Hospitals and Clinics during a 2-year period beginning in 1997. A review of the symptoms, physical findings, and radiographic abnormalities is presented. Treatment options, varying from muscle relaxants to surgical fusion, are discussed. A high index of suspicion in evaluating children with a stiff neck or pain on attempted motion is essential in order to facilitate prompt diagnosis and appropriate intervention

Expression of Aquaporin 1 and 5 in the Developing Mouse Inner Ear and Audiovestibular Assessment of an Aqp5 Null Mutant

To examine the potential roles of aquaporins 1 and 5 (AQP1 and AQP5, respectively) in inner ear development and function, we defined their spatial and temporal expression patterns in the developing mouse inner ear and examined the morphologic and physiologic effects of loss of Aqp5 function. Standard in situ hybridization (ISH) and immunohistochemical (IHC) assays were used for expression studies with routine morphologic, behavioral, and physiologic assessments of hearing and balance in Aqp5 null mutant mice. AQP1 was first detected at embryonic day 10.5 (E10.5) in the otocyst but eventually localized to specific nonsensory portions of the inner ear and connective tissue cells surrounding the membranous labyrinth. AQP5 displayed specific cochlear expression, first detectable at E15.5 in the nonsensory epithelium and later restricted to the lateral wall of the cochlear duct near the spiral prominence. AQP5 expression continued through postnatal periods with a change of expression domain to the stria vascularis between postnatal day 7 (P7) and P14. By in situ hybridization and immunohistochemical techniques, subtle differences between transcript and protein expression patterns were noted for both AQP1 and 5. Although AQP5 is dynamically expressed in the developing mouse inner ear, adult Aqp5 knockout mice show normal hearing when tested and normal inner ear structural development. These results suggest redundant or alternative mechanisms that likely regulate water homeostasis in the developing and mature inner ear

Treatment of lymphangiomas with OK-432 (Picibanil) sclerotherapy: A prospective multi-institutional trial

Objective: To describe and to determine the robustness of our study evaluating the efficacy of OK-432 (Picibanil) as a therapeutic modality for lymphangiomas. Design and Setting: Prospective, randomized trial and parallel-case series at 13 US tertiary care referral centers. Subjects: Thirty patients diagnosed as having lymphangioma. Ages in 25 ranged from 6 months to 18 years. Twenty-nine had lesions located in the head-and-neck area. Intervention: Every patient received a 4-dose injection series of OK-432 scheduled 6 to 8 weeks apart unless a contraindication existed or a complete response was observed before completion of all injections. A control group was observed for 6 months. Outcome Measures: Successful outcome of therapy was defined as a complete or a substantial (\u3e60%) reduction in lymphangioma size as determined by calculated lesion volumes on computed tomographic or magnetic resonance imaging scans. Results: Overall, 19 (86%) of the 22 patients with predominantly macrocystic lymphangiomas had a successful outcome. Conclusions: OK-432 should be efficacious in the treatment of lymphangiomas. Our study design is well structured to clearly define the role of this treatment agent