Comparison of fluorescent SSCP and denaturing HPLC analysis with direct sequencing for mutation screening in hypertrophic cardiomyopathy.

The recent achievement of the human genome project has led to the identification of many disease genes in common hereditary conditions, in which patients and their relatives would benefit from genetic diagnosis. This has increased the need for simple, sensitive, and cost effective methods of mutation analysis. However, the “gold standard” of mutation analysis, direct sequencing, is still an expensive and labour intensive method for investigation of large genes, multi-allelic diseases, and large numbers of patients. Today the most frequently used methods for prescreening disease genes for mutations are single strand conformation polymorphism analysis (SSCP) and denaturing high performance liquid chromatography (DHPLC). Both methods accelerate specific diagnosis at the DNA level by limiting the need for direct sequencing to a few abnormal polymerase chain reaction (PCR) products identified in the prescreening procedure. The tecnique of SSCP analysis is based on the principle that changes in nucleic acid composition affect the conformation of single stranded DNA and thereby the mobility of the fragment when it is subjected to electrophoresis under nondenaturing conditions. 2 Abnormal conformers identified in the prescreening procedure are subsequently investigated for the presence of sequence variants by direct sequencing. The tecnique was initially developed for manual gel electrophoresis in which the mobility of amplified PCR products was visualised by incorporation of radiolabelled nucleotides or by silver staining after electrophoresis. Recently, SSCP has been developed for automated capillary electrophoresis (CE) using buffered polymer solution as the sieving matrix for electrophoretic separation and fluorescence as the method of detection. Fluorescent capillary electrophoresis SSCP (F-SSCP) has the advantage of limited “post-PCR” handling of samples, which are loaded and analysed automatically with the possibility of a high throughput. Also, the requirements for reagent and sample volumes are small. Previous studies have reported a high sensitivity and specificity of F-SSCP. However, the primary method of identifying mutations used to investigate the efficacy of mutation detection by F-SSCP is unclear in several reports. Some reports investigated the ability of F-SSCP to identify mutations initially disclosed by manual SSCP. Only a few of the previously reported test mutations were identified by direct sequencing or introduced in specific test DNA by site directed mutagenesis. The technique of DHPLC has been developed more recently and relies on different retention times of homoduplex and heteroduplex DNA on ion pair reverse phase chromatography. Partial heat denaturation of samples decreases the retention time of mismatched DNA fragments compared with their intact double stranded counterparts. The method has similar advantages to F-SSCP, that is, automation, no “post-PCR” handling of samples, high throughput, and low cost, not least because PCR products are amplified with unlabelled primers. Several studies have reported high sensitivity and specificity when compared to mutations identified by various SSCP analyses and direct sequencing. This study investigated the suitability of F-SSCP and DHPLC for detection of sequence variants in five disease genes associated with the phenotypic expression of the most common hereditary heart condition known as hypertrophic cardiomyopathy (HCM). The disease is characterised by unexplained thickening of the heart muscle and predominantly inherited as an autosomal dominant trait with a prevalence of about 1:500 in a young adult population. Initially, the five genes were subjected to direct sequencing in 100 patients and 78 different sequence variants were identified. Subsequently, F-SSCP analysis was developed for a 16 capillary electrophoresis machine (ABI Prism 3100 genetic analyser, Applied Biosystems) and the ability to detect abnormal conformers of sequence variants identified by direct sequencing was evaluated. Similarly, the ability of DHPLC (Wave 3500HT DNA fragment analysis system, Transgenomic) to detect the same sequence variants was investigated. Key points