NONRADIOACTIVE MULTIPLEX PCR SCREENING STRATEGY FOR THE SIMULTANEOUS DETECTION OF MULTIPLE LOW-DENSITY-LIPOPROTEIN RECEPTOR GENE-MUTATIONS

We have developed a rapid, nonradioactive screening test enabling the simultaneous analysis of three low-density lipoprotein receptor (LDLR) gene mutations (D154N, D206E, and V408M), which together account for familial hypercholesterolemia (FH) in similar to 90% of the South Afri can Afrikaner population. The assay is designed so that FH patients, n egative for these founder-related mutations (found in descendants of E uropean settlers), subsequently can be screened for unknown mutations in the mutation-rich exon 4 of the LDLR gene. Our screening assay cons ists of two steps: (1) multiplex allele-specific PCR amplification of exons 4 and 9, and (2) simultaneous analysis of single- and double-str and conformational polymorphisms in exon 4 by vertical electrophoresis on low crosslinked polyacrylamide gels. The simplicity, specificity, and versatility of the multiplex assay makes it an ideal system for ro utine screening of FH mutations in large population samples.