Universal primer quantitative fluorescent multiplex (UPQFM) PCR: a method to detect major and minor rearrangements of the low density lipoprotein receptor gene

A method based on quantitative fluorescent multiplex PCR has been developed to detect major rearrangements of the low density lipoprotein receptor gen e (LDLR) which account for similar to 5% of mutations. The method involves two PCR reactions; the first (P1) amplifies the selected exons using unique primer sequences tagged with newly designed universal primers, while the s econd (P2) amplifies the P1 amplicons using the universal primers. One of t he P2 universal primers is labelled with a fluorescent dye which is incorpo rated into the PCR products which are then electrophoresed on an AB1 DNA se quencer. The relative amounts of the amplified peak areas are determined an d compared to ratios obtained for DNA from four normal controls and known m ajor rearrangements. The multiplex set developed is based on LDLR exons 3, 5, 8, 14, and 17 and 86% of reported major rearrangements would be detectab le by this assay as well as any deletions and insertions of greater than 1 bp. The method was evaluated using DNA from 15 reported deletions and dupli cations which were all correctly identified. Two groups of UK patients with a clinical diagnosis of familial hypercholesterolaemia (FH) and where no m utation had been identified in LDLR or APOB (14 children and 12 adults) wer e screened for the presence of major LDLR rearrangements by this assay. Thr ee major rearrangements were detected and a 4 bp duplication was identified in a fourth patient. Since it avoids the problems associated with Southern blotting, this method will be useful for detecting gene rearrangements.