A total of 188 probands and 456 family members (parents and sibs) were collected and studied as part of the UK NCUS. The protocol of the study adhered to the provisions of the Declaration of Helsinki and had multicentre research ethics approval granted for recruitment through Moorfields Eye Hospital, Great Ormond Street Hospital (who both also approved the study), the support organisation Sense, or as self-referrals. Informed consent to the study was obtained from all participants.
Patients were classified as Usher type I (USH1), II (USH2), III (USH3) or atypical based on ophthalmologic, audiometric and vestibular tests. Control DNA cohorts consisted of 381 unrelated UK blood donors (European Collection of Cell Cultures, ECCAC), 48 CEPH control DNAs (Caucasian, Utah, USA), and 57 individuals of Pakistani origin (courtesy of Professor Eamonn R Maher, Birmingham, UK).
Ophthalmic examination was performed in all affected individuals to confirm the presence of RP and included best corrected visual acuities, slit lamp biomicroscopy, colour vision testing with Hardy-Rand-Rittler colour plates, and Goldmann perimetry using the V4e, II4e and I4e targets. Retinal imaging with digital colour fundus photography, optical coherence tomography (6mm scans centred on the fovea; Stratus OCT3; Carl Zeiss Meditec, Dublin, California, USA) and fundus autofluorescence (FAF) imaging (HRA, Heidelberg, Germany) was also performed. Pattern and full field electroretinograms (ERGs) were performed in some cases using international standards.24 (link)
25 (link)
Audiologic evaluation included pure tone audiometry, tympanometry, stapedial reflex measurement, transient evoked otoacoustic emission recordings, and auditory brain stem evoked response recording using standard protocol.26–31 Subjective pure tone air and bone conduction thresholds were determined at 0.25, 0.5, 1, 2, 4, and 8 kHz using a GSI 61 audiometer (Guymark, Cradley Heath, UK), TDH39 supra aural earphones (Sennheiser UK, Ltd, High Wycombe, UK), and the British Society of Audiology recommended procedure. Audiometric descriptors of mild, moderate, severe, and profound hearing loss were calculated according to the British Society of Audiology descriptors. Vestibular function was evaluated with infrared video nystagmography, a rotary chair system (Neurokinetics, Pittsburgh, Pennsylvania, USA), and vestibulo-ocular reflex responses.30 (link) Binaural bithermal caloric testing with water was undertaken using the British Society of Audiology recommended protocol (http://www.thebsa.org.uk/docs/RecPro/CTP.pdf),29 (link) and the departmental normative data for peak slow component velocity were used to determine normality. Canal paresis (>17%) and directional preponderance (>16%) were calculated according to Jongkees formulae,32 (link) and vestibular hypofunction was defined by total eye velocity <78°/s. All parameters were defined by departmental normative data. Bilateral horizontal semicircular canal function was assessed using sinusoidal (60° peak velocity and 0.05 Hz) and step rotation testing (acceleration, 0°–60°/s constant velocity in <1 s). A gain of either <0.23 in test or time constant of <8 s on impulsive rotation was considered vestibular hypofunction.