Mutations in the low density lipoprotein receptor gene of familial hypercholesterolemic patients detected by denaturing gradient gel electrophoresis and direct sequencing

Familial hypercholesterolemia (FH) results from mutations in the low density lipoprotein receptor (LDLR) gene. We applied denaturing gradient gel electrophoresis (DGGE) to screen for sequence variations in the coding and splice site consensus sequences of the LDLR gene. For amplification of each exon by the polymerase chain reaction (PCR), optimal pairs of primers were designed by the MELT 87 computer algorithm. To increase the sensitivity, an artificial GC-clamp was included in either the 5'- or the 3'-end of each fragment. DGGE screening of 32 apparently unrelated heterozygous FH patients revealed 16 unique different aberrant DGGE patterns in 27 patients, while in a group of 32 normal subjects 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 revealed the nature of the mutations: three nonsense, four splicing, two frameshift, one silent, and six missense mutations. Six of the mutations have been previously reported, while ten are novel mutations. These results indicate that DGGE provides a reliable method for the detection of the presence of point mutations in the LDLR gene of FH patients, thereby facilitating the introduction of rapid DNA diagnosis for this common and genetically heterogeneous disorder