MUTATIONS IN THE LOW-DENSITY-LIPOPROTEIN RECEPTOR GENE OF FAMILIAR HYPERCHOLESTEROLEMIC PATIENTS DETECTED BY DENATURING GRADIENT GEL-ELECTROPHORESIS AND DIRECT SEQUENCING

Familial hypercholesterolemia (FH) results from mutations in the low d ensity lipoprotein receptor (LDLR) gene. We applied denaturing gradien t gel electrophoresis (DGGE) to screen for sequence variations in the coding and splice site consensus sequences of the LDLR gene. For ampli fication of each exon by the polymerase chain reaction (PCR), optimal pairs of primers were designed by the MELT 87 computer algorithm. To i ncrease the sensitivity an artificial GC-clamp was included in either the 5'- or the 3'-end of each fragment. DGGE screening of 32 apparentl y unrelated heterozygous FH patients revealed 16 unique different aber rant DGGE patterns in 27 patients, while in a group of 32 normal subje cts none of these DGGE patterns could be observed, suggesting that the aberrant patterns represent disease-causing mutations. Interestingly, 16 out of 27 patients showed an aberrant DGGE pattern in the part of the gene encoding the ligand binding domain (exons 2-6). Direct solid- phase sequencing of the corresponding exon-specific PCR products revea led the nature of the mutations: three nonsense, four splicing, two fr ameshift, one silent, and six missense mutations. Six of the mutations have been previously reported, while ten are novel mutations. These r esults indicate that DGGE provides a reliable method for the detection of the presence of point mutations in the LDLR gene of FH patients, t hereby facilitating the introduction of rapid DNA diagnosis for this c ommon and genetically heterogeneous disorder.