Genetic Testing of Korean Familial Hypercholesterolemia Using Whole-Exome Sequencing

<div><p>Familial hypercholesterolemia (FH) is a genetic disorder with an increased risk of early-onset coronary artery disease. Although some clinically diagnosed FH cases are caused by mutations in <i>LDLR</i>, <i>APOB</i>, or <i>PCSK9</i>, mutation detection rates and profiles can vary across ethnic groups. In this study, we aimed to provide insight into the spectrum of FH-causing mutations in Koreans. Among 136 patients referred for FH, 69 who met Simon Broome criteria with definite family history were enrolled. By whole-exome sequencing (WES) analysis, we confirmed that the 3 known FH-related genes accounted for genetic causes in 23 patients (33.3%). A substantial portion of the mutations (19 of 23 patients, 82.6%) resulted from 17 mutations and 2 copy number deletions in <i>LDLR</i> gene. Two mutations each in the <i>APOB</i> and <i>PCSK9</i> genes were verified. Of these anomalies, two frameshift deletions in <i>LDLR</i> and one mutation in <i>PCSK9 </i>were identified as novel causative mutations. In particular, one novel mutation and copy number deletion were validated by co-segregation in their relatives. This study confirmed the utility of genetic diagnosis of FH through WES.</p></div

Known pathogenic mutations in three FH-linked genes (n = 69).

<p>*Nucleotide location number was assigned according to the low-density lipoprotein receptor (<i>LDLR</i>; NM_000527), apolipoprotein B (<i>APOB</i>; NM_000384), and proprotein convertase subtilisin/kexin type 9 (<i>PCSK9</i>; NM_174936) mRNA sequences.</p><p>^†A patient (P49) with p.R257W (homozygote) and p.D589N (homozygote).</p><p>^‡Screening the remaining cohort by Sanger sequencing identified three more patients with p.P685L.</p><p>Variants were characterized in published studies and validated in the present study by Sanger sequencing.</p><p>Known pathogenic mutations in three FH-linked genes (n = 69).</p

Pedigree analysis of a patient with <i>LDLR</i> p.D834Rfs/- mutation.

<p>(A) A simplified pedigree of the P05 family. The upper right arrow indicates the proband; squares indicate males, and circles indicate females. Open and filled symbols indicate unaffected and affected individuals, respectively. Asterisks indicate family members who underwent clinical examinations and molecular analyses. WT refers wild-type. (B) Clinical examination data and sequencing chromatograms. Vertical arrows indicate the mutation site. (C) Integrative Genomics Viewer screenshot of p.D834Rfs/-. Sequencing reads show that a single nucleotide substitution (G>C) and frameshift deletion (AT/-) occurred at the cis position.</p

Novel pathogenic mutations in three FH-linked genes (n = 69).

<p>*Nucleotide location number was assigned according to the low-density lipoprotein receptor (<i>LDLR</i>; NM_000527) and proprotein convertase subtilisin/kexin type 9 (<i>PCSK9</i>; NM_174936) mRNA sequences.</p><p>^†Prediction for frameshift mutations of <i>LDLR</i> is not available from the Polyphen-2 and SIFT algorithms and is not marked.</p><p>^‡Public databases include the 1000 Genomes Project, dbSNP135, and NHLBI GO Exome Sequencing Project.</p><p>^§The frameshift mutation changes the cysteine at position 109, as four nucleotides after the deletion compensate for the frameshift effect until threonine (108).</p><p>^||The replacement of nucleotides 2500 to 2502 (GAT) occurred by ‘C’ at the cis position.</p><p>^#The p.R215H (c.644G>A) is a gain-of-function mutation in the catalytic domain of <i>PCSK9</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126706#pone.0126706.ref022" target="_blank">22</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126706#pone.0126706.ref023" target="_blank">23</a>] Variants were validated by Sanger sequencing.</p><p>NA: Not available.</p><p>Novel pathogenic mutations in three FH-linked genes (n = 69).</p

Clinical characteristics of enrolled familial hypercholesterolemia patients.

<p>*Chi-square test or t-test was used where appropriate.</p><p>Values are mean ± standard deviation or n (%).</p><p>HDL: high-density lipoprotein; LDL: low-density lipoprotein; Mutation (-): No known or novel pathogenic mutations in three FH-linked genes (<i>LDLR</i>, <i>APOB</i>, <i>PCSK9</i>); Mutation (+): Known or novel pathogenic mutations in <i>LDLR</i>, <i>APOB</i>, or <i>PCSK9</i>.</p><p>Clinical characteristics of enrolled familial hypercholesterolemia patients.</p

Copy number variation (CNV) detection in <i>LDLR</i>.

<p>SVD-ZRPKM values were used to detect CNVs by the CoNIFER algorithm and were calculated by transforming reads per kilobase per million values into standardized z-scores, based on the mean and standard deviation across all analyzed exomes. (A) The SVD-ZRPKM regional plot of the P25 patient with a large copy number deletion in <i>LDLR</i>. (B) The SVD-ZRPKM regional plot of the P17 patient and family member (P17-F01) with an inherited copy number deletion in <i>LDLR</i>. Green and blue indicate SVD-ZRPKM values of P17 and P17-F01, respectively. Values are plotted based on P17. (C) Pedigree of the P17 patient with CNV. The upper right arrow indicates the proband; squares indicate males, and circles indicate females. Open and filled symbols indicate unaffected and affected individuals, respectively. Asterisks indicate family members who underwent clinical examinations and CNV analyses. (D) TaqMan Copy Number Assay for P25, P17, and family members of P17. Red indicates the assay for P25 by probe #1 within intron 5; blue indicates the assay for P17 and other members by probe #2 (overlapped from intron 10 to exon 11). The assay was performed in duplicate and repeated. Results were plotted by CopyCaller software v.2.0.</p