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Development of a genotyping Microarray for usher syndrome
  1. Frans PM Cremers (f.cremers{at}antrg.umcn.nl)
  1. Radboud University Nijmegen Medical Centre, Netherlands
    1. William J Kimberling (kimber{at}boystown.org)
    1. Boys Town National Research Hospital, United States
      1. Maigi Kulm (maigi{at}asperbio.com)
      1. AsperBiotech, Tartu, Estonia
        1. Arjan de Brouwer (a.debrouwer{at}antrg.umcn.nl)
        1. Radboud University Nijmegen Medical Centre, Netherlands
          1. Erwin van Wijk (e.vanwijk{at}antrg.umcn.nl)
          1. Radboud University Nijmegen Medical Centre, Netherlands
            1. Heleen te Brinke
            1. Radboud University Nijmegen Medical Centre, Netherlands
              1. Cor WRJ Cremers (c.cremers{at}antrg.umcn.nl)
              1. Radboud University Nijmegen Medical Centre, Netherlands
                1. Lies H Hoefsloot (l.hoefsloot{at}antrg.umcn.nl)
                1. Radboud University Nijmegen Medical Centre, Netherlands
                  1. Sandro Banfi (banfi{at}tigem.it)
                  1. TIGEM, Italy
                    1. Francesca Simonelli
                    1. Department of Ophthalmology, Naples, Italy
                      1. Johannes C Fleischhauer
                      1. Department of Ophthalmology, Zurich, Switzerland
                        1. Wolfgang Berger (berger{at}medgen.unizh.ch)
                        1. University of Zurich, Switzerland
                          1. Phil M Kelley
                          1. Boystown National Research Hospital, United States
                            1. Elene Haralambous
                            1. Institute of Child Health, United Kingdom
                              1. Maria Bitner-Glindzicz (m.bitner-glindzicz{at}ich.ucl.ac.uk)
                              1. Institute of Child Health, United Kingdom
                                1. Andrew R Webster
                                1. Insititute of Ophthalmology, United Kingdom
                                  1. Zubin Saihan
                                  1. Insititute of Ophthalmology, United Kingdom
                                    1. Elfride De Baere
                                    1. Ghent University, Belgium
                                      1. Bart P Leroy
                                      1. Ghent University, Belgium
                                        1. Giuliana Silvestri
                                        1. Queen's University Belfast, United Kingdom
                                          1. Gareth McKay
                                          1. Queen's University Belfast, United Kingdom
                                            1. Robert K Koenekoop
                                            1. McGill University Health Center, Canada
                                              1. Jose M Millan
                                              1. Hospital Universitario La Fe, Spain
                                                1. Thomas Rosenberg
                                                1. National Eye Clinic for the Visually Impaired, Denmark
                                                  1. Tarja Joensuu
                                                  1. University of Helsinki, Finland
                                                    1. Eeva-Marja Sankila
                                                    1. University of Helsinki, Finland
                                                      1. Dominique Weil
                                                      1. Institut Pasteur, France
                                                        1. Mike D Weston
                                                        1. Boystown National Research Hospital, United States
                                                          1. Bernd Wissinger
                                                          1. University Eye Hospital, Germany
                                                            1. Hannie Kremer (h.kremer{at}antrg.umcn.nl)
                                                            1. Radboud University Nijmegen Medical Centre, Netherlands

                                                              Abstract

                                                              Introduction: Usher syndrome, a combination of retinitis pigmentosa (RP) and sensorineural hearing loss with or without vestibular dysfunction, displays a high degree of clinical and genetic heterogeneity. Three clinical subtypes can be distinguished, based on the age of onset and severity of the hearing impairment, and the presence or absence of vestibular abnormalities. Thus far, 8 genes have been implicated, which together comprise 347 protein-coding exons. Therefore, sequence analysis and the most routinely used mutation scanning techniques are not cost-effective for molecular diagnostics of Usher syndrome. To improve DNA-diagnostics for patients with Usher syndrome, we developed a genotyping microarray based on the arrayed primer extension (APEX) method.

                                                              Methods: Allele-specific oligonucleotides corresponding to 298 Usher syndrome-associated sequence variants known to date, 76 of which are novel, were arrayed. The accuracy of the microarray was analysed using DNAs from 158 patients with known mutations; the efficiency of the microarray was analysed using DNAs from 370 novel European and American patients with Usher syndrome.

                                                              Results: Validation of the microarray yielded an accuracy of >98%. Among the novel patients, sequence variants were identified in 64/140 (46%) patients with Usher syndrome type I (USH1), 45/189 (24%) patients with Usher syndrome type II (USH2), 6/21 (29%) patients with Usher syndrome type III (USH3), and 6/20 (30%) patients with atypical Usher syndrome. The chip also identified two novel sequence variants, c.400C>T (p.R134X) in PCDH15 and c.1606T>C (p.C536S) in USH2A.

                                                              Discussion: The Usher genotyping microarray represents a versatile and affordable screening tool for Usher syndrome. Its efficiency will improve with the addition of novel sequence variants with minimal extra costs, making it a very useful first-pass screening tool.

                                                              • Usher syndrome
                                                              • arrayed primer extension
                                                              • mutation analysis
                                                              • retinitis pigmentosa
                                                              • sensorineural deafness

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