Mutations in the gene for low density lipoprotein receptor (LDL-R) lead to a disorder called familial hypercholesterolemia (FH). Affected individuals are characterized by increased levels of cholesterol in plasma, tendinous xanthomas, arcus lipoides corneae, and premature coronary heart disease. Up to the present, more than 300 different mutations affecting the structure and function of cell surface LDLR have been identified (Goldstein et al., 1995). These are spread across the entire length of the gene, and no hot spots have so far been found.
During the past year, we collected 52 samples of genomic DNA from unrelated clinically diagnosed heterozygous FH patients recruited from lipid clinics at Brno. At the beginning, we excluded eight patients in which apoB-100 (R3500Q) mutation was detected (Hansen et al., 1991). As of now, there is no information about LDL-R gene mutation spectrum in the population of the Czech Republic. Here, we describe three mutations found in the EGF precursor homology domain LDL-R gene. Two of these point mutations-R395W and K497E-are novel.
Two approaches were used to identify mutations: (1) Direct P664L mutation screening in exon 14, using PCR/RFLP technique. Cytosine to thymine change could be detected with PstI digestion (Soutar et al., 1989).
(2) Heteroduplex analysis of PCR products of exons 9 and 10. Abberant mobility bands were detected, and the underlying mutations were characterized by solid phase sequencing. Here, we detected novel mutations R395W and K497E. The point mutation R395W causes arginin (CGG) being exchanged for tryptophan (TGG), and it is connected with the loss of one of the MspI restriction sites. In the normal allele, digestion of the 223 bp fragment produces 137,56, and 30 bp restriction fragments, whereas in the mutant allele fragment 167 bp is observed.
The mutation K497E is a single nucleotide change, substituting lysin (AAG) for glutamic acid (GAG). After using the SeqAid 11 Programm for searching the restriction enzymes for mutated sequences, we found there was no target sequence for restriction endonucleases in the mutated allele.