Whatley SD, Woods L, Mason NG, Roberts AG, Badminton MN

Department of Medical Biochemistry and Immunology, University Hospital of Wales and School of Medicine, Cardiff University, Cardiff, UK

Identification of mutations in the FECH gene is necessary for the accurate predictive genetic counselling of some families with EPP. However, with current methods, FECH mutations are not detected in about 20% of families. In an attempt to increase the detection rate, we are assessing a three stage strategy for mutation analysis. Denaturing HPLC was used as a first line screening test. Each exon was amplified from genomic DNA and analysed without further purification. Positive and negative controls for each exon were included with each batch of samples. Shifts, in addition to those caused by known SNPs, were identified in 32 (74%) of 43 patients; all were confirmed as mutations by fluorescent sequencing. For patients in whom mutations were not identified, quantitative PCR was used to search for deletions that encompassed exons and were not detectable by sequencing. Exons 2-11 were amplified by PCR in a multiplex reaction, with incorporation of a fluorescent label and subsequent analysis by gene scanning.  Exon dosage was determined by comparison with internal controls. Finally, mutation negative samples were analysed by bi-directional sequencing using different primers from those used for dHPLC to avoid the frequent problem of primer site polymorphisms. Using either this strategy, or bi-directional automated sequencing alone, we have so far identified FECH mutations in 61(77%) of 79 patients with EPP, only one of whom had a deletion undetectable by direct sequencing of PCR-amplified genomic DNA.