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.