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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.
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