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Molecular diagnosis of usher syndrome: application of two different next generation sequencing-based procedures
Authors:Danilo Licastro  Margherita Mutarelli  Ivana Peluso  Kornelia Neveling  Nienke Wieskamp  Rossella Rispoli  Diego Vozzi  Emmanouil Athanasakis  Angela D'Eustacchio  Mariateresa Pizzo  Francesca D'Amico  Carmela Ziviello  Francesca Simonelli  Antonella Fabretto  Hans Scheffer  Paolo Gasparini  Sandro Banfi  Vincenzo Nigro
Affiliation:Cluster in Biomedicine (CBM) scrl - Genomics, Area Science Park, Basovizza, Trieste, Italy.
Abstract:Usher syndrome (USH) is a clinically and genetically heterogeneous disorder characterized by visual and hearing impairments. Clinically, it is subdivided into three subclasses with nine genes identified so far. In the present study, we investigated whether the currently available Next Generation Sequencing (NGS) technologies are already suitable for molecular diagnostics of USH. We analyzed a total of 12 patients, most of which were negative for previously described mutations in known USH genes upon primer extension-based microarray genotyping. We enriched the NGS template either by whole exome capture or by Long-PCR of the known USH genes. The main NGS sequencing platforms were used: SOLiD for whole exome sequencing, Illumina (Genome Analyzer II) and Roche 454 (GS FLX) for the Long-PCR sequencing. Long-PCR targeting was more efficient with up to 94% of USH gene regions displaying an overall coverage higher than 25×, whereas whole exome sequencing yielded a similar coverage for only 50% of those regions. Overall this integrated analysis led to the identification of 11 novel sequence variations in USH genes (2 homozygous and 9 heterozygous) out of 18 detected. However, at least two cases were not genetically solved. Our result highlights the current limitations in the diagnostic use of NGS for USH patients. The limit for whole exome sequencing is linked to the need of a strong coverage and to the correct interpretation of sequence variations with a non obvious, pathogenic role, whereas the targeted approach suffers from the high genetic heterogeneity of USH that may be also caused by the presence of additional causative genes yet to be identified.
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