Mutational Spectrum of LDLR and PCSK9 Genes Identified in Iranian Patients With Premature Coronary Artery Disease and Familial Hypercholesterolemia

Abstract

Familial hypercholesterolemia (FH) is a common, yet underdiagnosed, genetic disorder characterized by lifelong elevated low-density lipoprotein cholesterol levels, which can increase the risk of early-onset coronary artery disease (CAD). In the present study, we screened the nucleotide variations of the LDLR and PCSK9 genes, as well as a part of the APOB gene, in Iranian patients with FH and premature CAD to find the genetic cause of the disorder. Fifteen unrelated individuals with a clinical diagnosis of FH and premature CAD were recruited. Direct DNA sequencing was applied to screen the whole coding exons and exon-intron boundaries of the LDLR and PCSK9 genes and the main parts of their introns, together with exon 26 of the APOB gene. The pathogenicity of the identified mutations was investigated via either segregation analyses in the family or in silico predictive software. Six different point mutations (p.Cys148Tyr, p.Cys216Tyr, p.Cys302Trp, p.Cys338Trp, p.Leu479Gln, and p.G593Afs^∗72) in LDLR and a double mutation (p.Asp172His and p.Ala53Val) in both LDLR and PCSK9 genes were identified in seven families with clinically diagnosed FH (43%), whereas no pathogenic mutations were found in eight families with clinically diagnosed FH. This study is the first to identify 1 pathogenic mutation in the LDLR gene (c.1014C > G [p.Cys338Trp]) and to cosegregate it from the affected individual in the family. No mutations were found in the APOB gene, whereas several silent mutations/polymorphisms were identified in the LDLR and PCSK9 genes. Genetic testing and reports on nucleotide alterations in the Iranian population are still limited. Our findings not only further confirm the significant role of FH in the incidence of premature CAD but also enlarge the spectrum of LDLR and PCSK9 variations and exhibit the heterogeneity of FH in Iranians. In patients with no mutation in the examined genes, the disease could be begotten either by a polygenic cause or by gene defects occurring in other related genes and regions not targeted in this study.

Keywords: Familial Hypercholesterolemia; LDLR; PCSK9 (proprotein convertase subtilisin kexin type 9); loss-of-function; pre-mature CAD.

FIGURE 1

The genomic and protein organization of LDLR. (A) The location of identified pathogenic mutations demonstrated in the different exons of LDLR gene. The novel mutation presented in blue color. (B) The domain organization of LDLR protein, which corresponded to LDLR coding exons (with the common colors).

FIGURE 2

Sequencing analysis and pedigree of proband who had double mutations in LDLR and PCSK9 genes. (A) The homozygote and heterozygote nucleotide changes at c.514G > C in LDLR demonstrated in upper chromaogram and lower chromatogram, respectively. (B) The loss-of-function mutation of PCSK9 in homozygote and heterozygote states depicted correspondingly in upper panel and lower panel. (C) The genotype of proband and her family members for LDLR and PCSK9 nucleotide changes demonstrated, respectively, in black and dark blue in the pedigree. The family history of proband exhibited positive history of hypercholesterolemia and cardiovascular diseases and myocardial infarction. The mother and father of the proband are first cousins. MI, myocardial infarction; HC, hypercholesterolemia; CD, cardiovascular diseases; CABG, coronary artery bypass grafting.

FIGURE 3

The chromatogram and pedigree of patient carried the novel mutation in LDLR gene. (A) The identified c.1014C > G nucleotide change shown in a chromatogram. (B) The pedigree of proband demonstrated the positive family history of hypercholesterolemia and myocardial infarction. The sister of proband is also heterozygote for the mutation. MI, myocardial infarction; HC, hypercholesterolemia.