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.

Figure 1

Pedigrees of families of Patients 1, 2 and 3. Gray symbols stand for microscopic hematuria and or proteinuria. Black symbols stand for ESRD.

Figure 2

Flowchart illustrating the different steps to filter variations detected by 454 technology in a pilot study of three ATS patients. This approach allowed to identify pathogenic mutations and to indicate cut-off values useful for flagging false-positive results.

Figure 3

Patient 2 (no. 2740) mutation detection. (a) A screenshot from the GS Amplicon Variant Analyzer software showing the COL4A3 missense sequence variant c.3440C>T (p.Ser1147Phe). The upper panel corresponds to a histogram indicating the percentage of variations. In the lower panel, reads from different directions are displayed and the mutated base is shown between the two vertical blue lines. Near the variation, there is a polyT stretch that creates technical artifacts (Supplementary Table 2). (b) Sanger sequencing chromatograms showing the missense sequence variant c.3440C>T (p.Ser1147Phe) found in Patient 2 respect to a control sample. The color reproduction of this figure is available at the European Journal of Human Genetics online.

Figure 4

Patient 3 (no. 3017) mutation detection. (a) A screenshot from the GS Amplicon Variant Analyzer software showing the COL4A4 missense sequence variant c.5044C>T (p.Arg1682Trp). The upper panel corresponds to a histogram indicating the percentage of variations. In the lower panel, reads from different directions are displayed and the mutated base is shown between the two vertical blue lines. (b) Sanger sequencing chromatograms showing the missense sequence variant c.5044C>T (p.Arg1682Trp) found in Patient 3 respect to a control sample. The color reproduction of this figure is available at the European Journal of Human Genetics online.