title: Advances in Alport syndrome diagnosis using next-generation sequencing.
abstract: Alport syndrome (ATS) is a hereditary nephropathy often associated with sensorineural hypoacusis and ocular abnormalities. Mutations in the COL4A5 gene cause X-linked ATS. Mutations in COL4A4 and COL4A3 genes have been reported in both autosomal recessive and autosomal dominant ATS. The conventional mutation screening, performed by DHPLC and/or Sanger sequencing, is time-consuming and has relatively high costs because of the absence of hot spots and to the high number of exons per gene: 51 (COL4A5), 48 (COL4A4) and 52 (COL4A3). Several months are usually necessary to complete the diagnosis, especially in cases with less informative pedigrees. To overcome these limitations, we designed a next-generation sequencing (NGS) protocol enabling simultaneous detection of all possible variants in the three genes. We used a method coupling selective amplification to the 454 Roche DNA sequencing platform (Genome Sequencer junior). The application of this technology allowed us to identify the second mutation in two ATS patients (p.Ser1147Phe in COL4A3 and p.Arg1682Trp in COL4A4) and to reconsider the diagnosis of ATS in a third patient. This study, therefore, illustrates the successful application of NGS to mutation screening of Mendelian disorders with locus heterogeneity.
authors: Medical Genetics Section, Biotechnology Department, University of Siena, Siena, Italy.
authors: Rosangela
authors: R
authors: Artuso
authors: Chiara
authors: C
authors: Fallerini
authors: Laura
authors: L
authors: Dosa
authors: Francesca
authors: F
authors: Scionti
authors: Maurizio
authors: M
authors: Clementi
authors: Guido
authors: G
authors: Garosi
authors: Laura
authors: L
authors: Massella
authors: Maria Carmela
authors: MC
authors: Epistolato
authors: Roberta
authors: R
authors: Mancini
authors: Francesca
authors: F
authors: Mari
authors: Ilaria
authors: I
authors: Longo
authors: Francesca
authors: F
authors: Ariani
authors: Alessandra
authors: A
authors: Renieri
authors: Mirella
authors: M
authors: Bruttini
mesh_terms: D000293:Adolescent; D000328:Adult; D001324:Autoantigens / Q000235:genetics*; D001483:Base Sequence; D002648:Child; D024141:Collagen Type IV / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D005820:Genetic Testing / Q000379:methods*; D015894:Genome, Human; D005838:Genotype; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D009394:Nephritis, Hereditary / Q000175:diagnosis* / Q000235:genetics; D010375:Pedigree; D010865:Pilot Projects
chemical_list: D001324:Autoantigens; D024141:Collagen Type IV; C057112:type IV collagen alpha3 chain