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

Abstract

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.

Molecular characterization of familial hypercholesterolemia in Spain: Identification of 39 novel and 77 recurrent mutations in LDLR

“…In this large cohort of Spanish patients with the clinical diagnosis of FH, the SSCP analysis of the promoter region and the 18 exons encompassing the LDLR protein coding and splice site consensus sequences revealed, in addition to 9 common polymorphisms (Jensen et al, 1996), aberrant SSCP patterns in all exons except exon 18. DNA sequencing and restriction analysis of the corresponding amplified exons revealed 116 different mutations causing these aberrant patterns.…”

“…Genomic DNA was extracted from peripheral leukocytes using standard methods. The promoter region, the translated exon sequences, and the exon-intron boundaries of the LDLR gene were individually amplified by PCR, and subsequently analyzed by SSCP (Jensen et al, 1996).…”

“…The haplotypes of FH subjects were determined using 8 polymorphisms in the LDLR gene. These polymorphisms were detected by PCR amplification and restriction enzyme digestion (Hobbs et al, 1990;Soutar, 1991;Saint-Jore et al, 1993;Jensen et al, 1996) (Table 2).…”

Mutation analysis in 36 unrelated Spanish subjects with familial hypercholesterolemia: Identification of 3 novel mutations in the LDL receptor gene

“…Genomic DNA was extracted from peripheral leukocytes of FH patients using standard methods. The promoter region and the 18 exons of the LDLR gene were amplified by the polymerase chain reaction (PCR) using the oligonucleotides described by Jensen et al (1996), but labelled at 5' position with Cy5 TM , and subsequently analyzed by SSCP. The PCR and SSCP conditions used were previously described .…”

Multiplex ligation‐dependent probe amplification analysis to screen for deletions and duplications of the LDLR gene in patients with familial hypercholesterolaemia

“…Large rearrangements such as deletions or duplications of one or more exons are thought to comprise around 5% of mutations in LDLR gene in the UK (6). However, this figure could be an underestimate as screening programmes have tended to focus on screening for point mutations by methods such as single-strand conformation polymorphism (SSCP) analysis (7), denaturing high-performance liquid chromatography (dHPLC) electrophoresis (8), and more recently by direct sequence analysis (9). The breakpoints of the various rearrangements identified span the LDLR gene, but the majority are located in introns 1-8 and 12 through the 3#-UTR (5).…”