High sensitivity of the single-strand conformation polymorphism method for detecting sequence variations in the low-density lipoprotein receptor gene validated by DNA sequencing.

We designed oligonucleotide primer pairs to amplify the promoter region, the translated exon sequences, and the flanking intron sequences of all 18 exons of the LDL receptor gene to compare the ability of the PCR single-strand conformation polymorphism (PCR-SSCP) method with semiautomated solid-phase genomic DNA sequencing to detect sequence variations. In 20 apparently unrelated Danish patients with a clinical diagnosis of heterozygous familial hypercholesterolemia (FH), we identified 13 different mutations in the LDL receptor gene: two silent (C331C, N494 N); five missense (W66G, E119K, T383P, W556S, T7051); one nonsense (W23X); three splice-site (313 + 1G-->A, 1061-8T-->C, 1846-1G-->A); and two frameshift (335del10, 1650delG) mutations. Four of these mutations, N494 N, T383P, 1061-8T-->C, and W556S, have not been reported earlier. The pathogenicity of the T383P, 1061-8T-->C, and W556S mutations remains to be established by in vitro mutagenesis and transfection studies. One patient had three mutations (335del10, 1061-8T-->C, and T705I) on the same allele. Further, nine well-known polymorphisms were detectable with this methodological setup. Direct DNA sequencing of the PCR products used for the SSCP analysis did not reveal any sequence variations not detected by the PCR-SSCP method. In two patients we did not detect any mutation by either method. We conclude that the PCR-SSCP analysis, performed as described here, is as sensitive and efficient as DNA sequencing in the ability to identify the sequence variations in the LDL receptor gene of the patients with heterozygous FH of this study.