Abstract
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.
Highlights
Mutations in the ABCA1 gene are the cause of familial high density lipoprotein deficiency (FHD)
We took advantage of all of the sequence variants we identified in this gene during the genotyping of subjects with Tangier disease (TD) and FHD
To compare the results of our previous sequencing work with those obtained with denaturing high-performance liquid chromatography (DHPLC), we used the same primers previously used for sequencing
Summary
Mutations in the ABCA1 gene are the cause of familial high density lipoprotein deficiency (FHD). Well-established techniques for mutation detection include relatively simple methods [such as single-strand conformation polymorphism (SSCP)] as well as more complex procedures [denaturing gradient gel electrophoresis (DGGE) or enzymatic and chemical cleavage] [1] Some of these methods, such as SSCP, have low sensitivity, whereas others, such as DGGE, are time-consuming, expensive, and difficult to apply to a large number of samples or to be automated [1]. Denaturing high-performance liquid chromatography (DHPLC) is a relatively novel technique that appears to meet the requirements specified above for large-scale screening of sequence variants. This technique is based on the detection of heteroduplexes in PCR products by ion-pair reversed-phase liquid chromatography under partial denaturing conditions within an acetonitrile gradient. The major advantages of this method are the use of automated instrumentation and the speed of analysis [2]
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