J Med Genet 2000;37:272-280 ( April )

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

Karen E Heath^{a b}, Ian N M Day^{* a}, Steve E Humphries^a

^a Centre for Cardiovascular Genetics, The Rayne Institute, University College Medical School, 5 University Street, London WC1E 6JJ, UK, ^b Unit of Clinical Molecular Genetics, Camelia Botnar Laboratories, Great Ormond Street Hospital for Children, Great Ormond Street, London WC1N 3JH, UK

Correspondence to: Dr Humphries, rmhaseh{at}ucl.ac.uk

Revised version received 28 October 1999; Accepted for publication 24 November 1999

A method based on quantitative fluorescent multiplex PCR has been developed to detect major rearrangements of the low density lipoprotein receptor gene (LDLR) which account for ~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 second (P2) amplifies the P1 amplicons using the universal primers. One of the P2 universal primers is labelled with a fluorescent dye which is incorporated into the PCR products which are then electrophoresed on an ABI DNA sequencer. The relative amounts of the amplified peak areas are determined and compared to ratios obtained for DNA from four normal controls and known major rearrangements. The multiplex set developed is based on LDLR exons 3, 5, 8, 14, and 17 and 86% of reported major rearrangements would be detectable 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 duplications which were all correctly identified. Two groups of UK patients with a clinical diagnosis of familial hypercholesterolaemia (FH) and where no mutation had been identified in LDLR or APOB (14 children and 42 adults) were screened for the presence of major LDLR rearrangements by this assay. Three 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.


Keywords: familial hypercholesterolaemia; LDLR; major rearrangements; universal primer quantitative fluorescent multiplex PCR (UPQFM-PCR)

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^* Present address: Wessex Human Genetics Institute, Duthie Building (808), Southampton University Hospitals NHS Trust, Tremona Road, Southampton SO16 6YD, UK

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© 2000 by J Med Genet
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