title: Molecular diagnosis of Usher syndrome: application of two different next generation sequencing-based procedures.
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.
authors.affiliation: Cluster in Biomedicine scrl - Genomics, Area Science Park, Basovizza, Trieste, Italy.
authors.forename: Danilo
authors.initials: D
authors.lastname: Licastro
authors.forename: Margherita
authors.initials: M
authors.lastname: Mutarelli
authors.forename: Ivana
authors.initials: I
authors.lastname: Peluso
authors.forename: Kornelia
authors.initials: K
authors.lastname: Neveling
authors.forename: Nienke
authors.initials: N
authors.lastname: Wieskamp
authors.forename: Rossella
authors.initials: R
authors.lastname: Rispoli
authors.forename: Diego
authors.initials: D
authors.lastname: Vozzi
authors.forename: Emmanouil
authors.initials: E
authors.lastname: Athanasakis
authors.forename: Angela
authors.initials: A
authors.lastname: D'Eustacchio
authors.forename: Mariateresa
authors.initials: M
authors.lastname: Pizzo
authors.forename: Francesca
authors.initials: F
authors.lastname: D'Amico
authors.forename: Carmela
authors.initials: C
authors.lastname: Ziviello
authors.forename: Francesca
authors.initials: F
authors.lastname: Simonelli
authors.forename: Antonella
authors.initials: A
authors.lastname: Fabretto
authors.forename: Hans
authors.initials: H
authors.lastname: Scheffer
authors.forename: Paolo
authors.initials: P
authors.lastname: Gasparini
authors.forename: Sandro
authors.initials: S
authors.lastname: Banfi
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Nigro
mesh_terms: D002675:Child, Preschool; D059472:Exome / Q000235:genetics; D015894:Genome, Human / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D010865:Pilot Projects; D017422:Sequence Analysis, DNA / Q000379:methods*; D052245:Usher Syndromes / Q000175:diagnosis* / Q000235:genetics*