Development of a universal chemiluminometric genotyping method for high-throughput detection of 7 LDLR gene mutations in Greek population.

OBJECTIVES: Familial hypercholesterolemia (FH) is caused by mutations in the LDL receptor (LDLR) gene. We report the application of a universal method with high allele discrimination properties to the simultaneous genotyping of 7 LDLR mutations in Greeks, in dry-reagent format. DESIGN AND METHODS: We genotyped mutations C858A, C939A, G1285A, T1352C, G1646A, G1775A, C/T81G. Unpurified amplicons from a multiplex PCR that produced fragments encompassing all 7 mutations were subjected to probe extension reactions in the presence of fluorescein-modified dCTP, and a microtiter well-based assay of extension products with a peroxidase-antifluorescein conjugate and a chemiluminogenic substrate. We used lyophilized dry reagents and assigned genotypes by the signal ratio of normal-to-mutant-specific probe.
RESULTS: We standardized the method and optimised all steps for specificity. The method was validated by genotyping blindly 119 (833 genotypings). Results were fully concordant with other methods used as standards.
CONCLUSIONS: This method is accurate, simple, rapid and robust. The microtiter well format allows genotyping of a large number of samples in parallel for several mutations.