Rapid detection by fluorescent multiplex PCR of exon deletions and duplications in the C1 inhibitor gene of hereditary angioedema patients

Hereditary angioedema (HAE) is due to a variety of defects in the C1 inhibi tor gene (C1NH gene), including approximately 20% of partial deletions/dupl ications whose boundaries are usually within Alu repeats. To ensure complet e molecular characterization of C1 inhibitor deficiencies a fluorescent mul tiplex assay was constructed to amplify simultaneously five exons of C1NH a nd an exon of the BRCA1 gene. PCR protocols were optimized for these amplic ons (size range between 300 and 700 bp). Forward and reverse chimeric prime rs that carry strand-specific 5' tags of 16 nucleotides were used to ensure similar levels of PCR products for each amplicon in the multiplex. Data we re analyzed by superposing fluorescent profiles of test and control DNA and by visually comparing the normalized peak levels of corresponding amplicon s, rather than by calculating the ratios of peak areas. Tests on a collecti on of known defects, including five different Alu-mediated deletions and a partial duplication have validated this approach. In a study of 19 sporadic cases of HAE, of which four had failed to reveal mutations upon screening all exons by fluorescent chemical cleavage, three de novo deletions were di agnosed by using this multiplex PCR approach: a deletion of exon 4, a delet ion of exons 5 and 6, and an apparently complete gene deletion. Besides bei ng suitable for the initial DNA screening of the C1NH gene in HAE patients prior to screening for point mutations, this method can be easily adapted t o complex genes for the screening of rearrangements. Hum Mutat 17:61-70, 20 01. (C) 2001 Wiley Liss, Inc.