High-resolution melting analysis for detection of familial ligand-defective apolipoprotein B-100 mutations

Ann Clin Biochem 2008;45:170-176
© 2008 Association for Clinical Biochemistry

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Original Article

Khemanganee E Liyanage 1 *,
Amanda J Hooper 2 *,
Joep C Defesche 3,
John R Burnett 2 4 and
Frank M van Bockxmeer 1 2 

1 School of Surgery and Pathology, University of Western Australia, Perth, Australia;
2 Department of Core Clinical Pathology and Biochemistry, PathWest Laboratory Medicine WA, Royal Perth Hospital, Perth, Australia;
3 Department of Vascular Medicine, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands;
4 School of Medicine and Pharmacology, University of Western Australia, Perth, Australia

Corresponding author: Associate Prof Frank van Bockxmeer. Email: Frank.VB{at}health.wa.gov.au

Background: Familial ligand-defective apolipoprotein B-100 (FDB) is characterizedby elevated plasma concentrations of LDL-cholesterol and apolipoprotein(apo) B, normal triglyceride and HDL-cholesterol levels, thepresence of tendon xanthomas, and premature coronary arterydisease. FDB cannot be clinically distinguished from heterozygousLDL-receptor-defective familial hypercholesterolaemia (FH) withoutgenetic testing.

Methods: Amplicons in exon 26 and exon 29 of the APOB gene were screenedfor established genetic variants including mutations and polymorphismsusing high-resolution melting analysis. Six novel variants associatedwith FDB in hypercholesterolaemic Dutch patients (S3476L, S3488G,Y3533C, T3540M, I4350T, G4368D) were also studied.

Results: All positive controls, a total of 10 mutations in exon 26 andfour mutations in exon 29, were readily detectable by meltingcurve analysis. In addition, a patient previously not knownto be heterozygous for the H3543Y mutation was identified ina screen of hypercholesterolaemic subjects. The method was validatedby comparison of high-resolution melting analysis with DNA sequencedata in a ‘blinded’ manner in 35 consecutive patientsattending a lipid disorders clinic. These patients were classifiedas ‘definite FH’ by the Dutch Lipid Clinic Networkcriteria. Five patients were found to be heterozygous for theR3500Q and one for H3543Y.

Conclusions: We have established a novel, robust method of FDB mutation detectionusing high-resolution melting analysis in conjunction with DNAsequencing. Compared with existing methods it is not only morecost-effective, but is also capable of detecting new sequencechanges and will have importance in cascade screening of affectedsubjects.

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