Efficient detection of Alport syndrome COL4A5 mutations with multiplex genomic PCR-SSCP

We have performed effective mutation screening of COL4A5 with a new method of direct, multiplex genomic amplification that employs a single buffer con dition and PCR profile. Application of the method to a consecutive series o f 46 United States patients with diverse indications of Alport syndrome res ulted in detection of mutations in 31 cases and of five previously unreport ed polymorphisms. With a correction for the presence of cases that are not likely to be due to changes at the COL4A5 locus, the mutation detection sen sitivity is greater than 79%. The test examines 52 segments, including the COL4A6/COL4A5 intergenic promoter region, all 51 of the previously recogniz ed exons and two newly detected exons between exons 41 and 42 that encode a n alternatively spliced mRNA segment. New genomic sequence information was generated and used to design primer pairs that span substantial intron sequ ences on each side of all 53 exons, For SSCP screening, 16 multiplex PCR co mbinations (15 4-plex and 1 3-plex) were used to provide complete, partiall y redundant coverage of the gene. The selected combinations allow clear res olution of products from each segment using various SSCP gel formulations. One of the 29 different mutations detected initially seemed to be a missens e change in exon 32 but was found to cause exon skipping, Another missense variant may mark a novel functional site located in the collagenous domain. (C) 2001 Wiley-Liss, Inc.