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Papers In Press, published online ahead of print April 1, 2005
J. Lipid Res., doi:10.1194/jlr.D400038-JLR200

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Journal of Lipid Research, Vol. 46, 817-822, April 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Methods

Denaturing high-performance liquid chromatography in the detection of ABCA1 gene mutations in familial HDL deficiency

Tommaso Fasano^*, Letizia Bocchi^*, Livia Pisciotta, Stefano Bertolini and Sebastiano Calandra^1,*

^* Department of Biomedical Sciences, University of Modena & Reggio Emilia, Modena, Italy
Department of Internal Medicine, University of Genova, Genova, Italy

The online version of this article (available at http://www.jlr.org) contains an additional seven tables and two figures.

Published, JLR Papers in Press, February 1, 2005. DOI 10.1194/jlr.D400038-JLR200

¹ To whom correspondence should be addressed. e-mail: sebcal{at}unimore.it

Mutations in the ABCA1 gene are the cause of familial high density lipoprotein deficiency (FHD). Because these mutations are spread over the entire gene, their detection requires the sequencing of all 50 exons. The aim of this study was to validate denaturing high-performance liquid chromatography (DHPLC) in mutation detection as an alternative to systematic sequencing. Exons of the ABCA1 gene were amplified using primers employed for sequencing. Temperatures for DHPLC were deducted from a software and empirically defined for each amplicon. To assess DHPLC reliability, we tested 30 sequence variants found in FHD patients and controls. Combined DHPLC and sequencing was applied to the genotyping of new FHD patients. Most of the amplicons required from two to five temperature conditions to obtain partially denatured DNA over the entire amplicon length. Twenty-nine of the variants found by sequencing were detected by DHPLC (97% sensitivity). The detection of the last variant (in exon 40) required different primers and amplification conditions. DHPLC and sequencing analysis of new FHD patients revealed that all amplicons showing a heteroduplex DHPLC profile contained sequence variants. No variants were detected in amplicons with a homoduplex profile.

DHPLC is a sensitive and reliable method for the detection of ABCA1 gene mutations.

Supplementary key words high density lipoprotein • ATP binding cassette transporter A1 • heteroduplexes • sequence variants • primary hypoalphalipoproteinemia

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