In the course of investigations of familial coronary artery disease in Hokkaido, the northern island of Japan, we identified five families in which multiple members showed elevated plasma levels of low-density lipoprotein (LDL) cholesterol. To determine the genetic etiology of their lipoprotein abnormalities, we screened DNA samples from these families for mutations in all 18 exons and the exon-intron boundaries of the LDL receptor (LDLR) gene. Novel point mutations were identified in each family: (1) a C-to-A transversion at nucleotide 285, causing a nonsense mutation at codon 74, in eight members of family A; (2) a G-to-A transition at nucleotide 1136, causing substitution of Tyr for Cys at codon 358, in six members of family B; (3) a C-to-T transition at nucleotide 1822, causing substitution of Ser for Pro at codon 587, in five members of family C; (4) a one-base insertion of G to a five-G stretch at nucleotides 1774-1778 (codons 571-572), causing a frameshift, in six members of family D; and (5) a one-base deletion of T at nucleotide 1963-1964 (codon 634), causing a frameshift, in three members of family E. Through the molecular genetic approach a total of 28 individuals in these families were diagnosed unequivocally as heterozygous for the respective LDLR mutations. This method also helped us to diagnose familial hypercholesterolemia, or to exclude from carrier status, 11 children with borderline high cholesterol levels.

Plasma lipid and lipoprotein levels reflect in part the influence of relevant genetic loci. Defects at some of these loci account for specific types of dyslipoproteinemia occurring with regularity among family members. In the course of familial investigations of coronary artery disease, we identified an family in which several members were affected with elevated low density lipoprotein (LDL) cholesterol levels. To study the genetic defects responsible for plasma lipoprotein abnormality in this pedigree, we developed a simple method for genotyping a single base substitution that does not affect a restriction recognition enzyme site in exon 10 of the LDL receptor gene. Using our mismatched PCR method, this G->A substitution at nucleotide 1413 could be genotyped in the form of a biallelic restriction fragment length polymorphism (RFLP) after digestion with restriction enzyme Hpa II. Linkage analysis using this molecular method demonstrated that the defect at the LDL receptor locus is responsible for elevated LDL cholesterol phenotype observed in this family by segregation of defective alleles at the LDL receptor locus with the disease (peak decimal logarithm of odds score>3.0).

SUMMARYIn the course of investigations of coronary artery disease in Utah, we identified a family whose proband showed elevated plasma levels of LDL cholesterol. To determine the genetic etiology of the lipoprotein abnormalities, we screened DNA samples for mutations in all 18 exons and the exon-intron boundaries of the low-density lipoprotein (LDL) receptor gene. Novel point mutations were identified in the proband: a T-to-A transversion at nucleotide position 223, causing substitution of Ser for Cvs at codon 54 in exon 3 of the receptor gene. This amino acid replacement would disrupt one of the disulfide bonds necessary for maintenance of the secondary structure of the repeat at the N-terminal of the receptor, prevent correct folding of the receptor, and result in defective intracellular transport of the receptor. (Jpn Heart j 1998; 39: 785-789)