Submitted on December 7, 2004
Revised on January 11, 2005
Accepted on January 19, 2005
Denaturing high-performance liquid chromatography (DHPLC) in the detection of mutations of ABCA1 gene in familial HDL deficiency
Tommaso Fasano, Letizia Bocchi, Livia Pisciotta, Stefano Bertolini, and Sebastiano Calandra
Biomedical Sciences, University of Modena and Reggio Emilia, Modena I-41100
Corresponding Author: sebcal{at}unimore.it
Mutations in ABCA1 gene are one of the genetic causes of familial high density lipoprotein deficiency (FHD). Since these mutations are spread over the entire gene, their detection requires the sequencing of all 50 exons of the gene, an expensive and time- consuming procedure. Aim of this study was to validate the use of denaturing high-performance liquid chromatography (DHPLC) in the detection of ABCA1 mutations as an alternative to systematic sequencing. All exons of ABCA1 gene were amplified by PCR using primers previously employed for sequencing. The temperatures for DHPLC analysis were deducted from a specific software and empirically defined for each amplicon. To assess reliability of DHPLC we tested 30 sequence variants previously found in FHD patients and controls. In addition combined DHPLC and sequencing was applied to the genotyping of three new FHD patients. Most of the amplicons required from a minimum of two to a maximum of five temperature conditions to obtain partially denatured DNA over the whole length of the amplicon. Twenty-nine of the variants previously found by sequencing were detected by DHPLC (97% sensitivity). The detection of the last variant (in exon 40) required different primers and PCR amplification conditions. The combined DHPLC and sequencing analysis of three new FHD patients revealed that all amplicons, showing heteroduplex DHPLC profile, contained sequence variants. No sequence variants were detected in the amplicons with an homoduplex DHPLC profile. DHPLC is a highly sensitive and reliable method for detection of ABCA1 gene sequence variants.
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