Familial hypercholesterolemia (FH), a frequent monogenic condition complicated by premature cardiovascular disease, is characterized by high allelic heterogeneity at the low-density lipoprotein receptor ( LDLR) locus. Despite more than a decade of genetic testing, knowledge about intronic disease-causing mutations has remained limited because of lack of available genomic sequences. Based on the finding from bioinformatic analysis that Alu repeats represent 85% of LDLR intronic sequences outside exon-intron junctions, we designed a strategy to improve the exploration of genomic regions in the vicinity of exons in 110 FH subjects from an admixed population. In the first group of 42 patients of negative mutation carriers, as previously established by former screening strategies (denaturing gradient gel electrophoresis, DNA sequencing with former primers overlapping splice-sites, Southern Blotting), about half ( n=22) were found to be carriers of at least one heterozygous mutation. Among a second group of 68 newly recruited patients, 27% of mutation carriers ( n=37) had a splicing regulatory mutation. Overall, out of the 54 mutations identified, 13 were intronic, and 18 were novel, out of which nearly half were intronic. Two novel intronic mutations (IVS8-10G-->A within the polypyrimidine tract and IVS7+10G-->A downstream of donor site) might create potential aberrant splice sites according to neural-network computed estimation, contrary to 31 common single nucleotide variations also identified at exon-intron junctions. This new strategy of detecting the most likely disease-causing LDLR mutations outside of Alu-rich genomic regions reveals that intronic mutations may have a greater impact than previously reported on the molecular basis of FH.

We used the denaturing gradient gel electrophoresis (DGGE) method to define mutations in the promoter region, the 18 exons, and their flanking intronic sequences of the low-density lipoprotein (LDL) receptor gene LDLR, causing familial hypercholesterolemia (FH) phenotype in 100 German and in 100 Greek hypercholesterolemic individuals. In addition, we tested all patients for the presence of mutations in codons 3456-3553 of the gene encoding apolipoprotein B-100 (APOB). Twenty-six aberrant DGGE patterns were identified and subsequently directly sequenced. In LDLR, two novel missense mutations (c.1957G>T/p.V653F, c.647 G>A/p.C216Y) and one novel homozygous base substitution c.1-156 C>T in the repeat 2 of the promoter region were identified among German FH patients; one novel splice site c.1060+10C>G was identified among Greek FH patients. One of the German FH patients was a carrier for the mutations c.1171G>A/p.A391T and p.V653F, and two of the Greek FH patients were compound heterozygotes for the mutations c.1150C>T/p.Q384X and c.1158C>G/p.D386E. Two German FH patients carried the mutation p.R3500Q within APOB. Comparing the mutations within the LDLR gene of the two European FH populations, the German population seems to be more heterogeneous than the Greek cohort. Further studies in progress are trying to elucidate the responsiveness to drug therapy in association with LDLR genotype and the nutritional habits of the two FH populations.