Lifelong Reduction of LDL-Cholesterol Related to a Common Variant in the LDL-Receptor Gene Decreases the Risk of Coronary Artery Disease—A Mendelian Randomisation Study

Rare mutations of the low-density lipoprotein receptor gene (LDLR) cause familial hypercholesterolemia, which increases the risk for coronary artery disease (CAD). Less is known about the implications of common genetic variation in the LDLR gene regarding the variability of cholesterol levels and risk of CAD.Imputed genotype data at the LDLR locus on 1 644 individuals of a population-based sample were explored for association with LDL-C level. Replication of association with LDL-C level was sought for the most significant single nucleotide polymorphism (SNP) within the LDLR gene in three European samples comprising 6 642 adults and 533 children. Association of this SNP with CAD was examined in six case-control studies involving more than 15 000 individuals.Each copy of the minor T allele of SNP rs2228671 within LDLR (frequency 11%) was related to a decrease of LDL-C levels by 0.19 mmol/L (95% confidence interval (CI) [0.13-0.24] mmol/L, p = 1.5x10(-10)). This association with LDL-C was uniformly found in children, men, and women of all samples studied. In parallel, the T allele of rs2228671 was associated with a significantly lower risk of CAD (Odds Ratio per copy of the T allele: 0.82, 95% CI [0.76-0.89], p = 2.1x10(-7)). Adjustment for LDL-C levels by logistic regression or Mendelian Randomisation models abolished the significant association between rs2228671 with CAD completely, indicating a functional link between the genetic variant at the LDLR gene locus, change in LDL-C and risk of CAD.A common variant at the LDLR gene locus affects LDL-C levels and, thereby, the risk for CAD

Dysfunctional nitric oxide signalling increases risk of myocardial infarction

Myocardial infarction, a leading cause of death intheWesternworld(1), usually occurs when the fibrous cap overlying an atherosclerotic plaque in a coronary artery ruptures. The resulting exposure of blood to the atherosclerotic material then triggers thrombus formation, which occludes the artery(2). The importance of genetic predisposition to coronary artery disease and myocardial infarction is best documented by the predictive value of a positive family history(3). Nextgeneration sequencing in families with several affected individuals has revolutionized mutation identification(4). Here we report the segregation of two private, heterozygous mutations in two functionally relatedgenes, GUCY1A3 (p.Leu163Phefs*24) andCCT7 (p.Ser525Leu), in an extended myocardial infarction family. GUCY1A3 encodes the alpha 1 subunit of soluble guanylyl cyclase (alpha 1-sGC)(5), and CCT7 encodes CCT eta, a member of the tailless complex polypeptide 1 ring complex(6), which, among other functions, stabilizes soluble guanylyl cyclase. After stimulation with nitric oxide, soluble guanylyl cyclase generates cGMP, which induces vasodilation and inhibits platelet activation(7). Wedemonstratein vitro that mutations inbothGUCY1A3 and CCT7 severely reduce alpha 1-sGC as well as beta 1-sGC protein content, and impair soluble guanylyl cyclase activity. Moreover, platelets from digenic mutation carriers contained less soluble guanylyl cyclase protein and consequently displayed reduced nitric-oxideinduced cGMP formation. Mice deficient in alpha 1-sGC protein displayed accelerated thrombus formation in themicrocirculation after local trauma. Starting with a severely affected family, we have identified a link between impaired soluble-guanylyl-cyclase-dependent nitric oxide signalling and myocardial infarction risk, possibly through accelerated thrombus formation. Reversing this defect may provide a new therapeutic target for reducing the risk of myocardial infarction

Abstract 19944: A PDE5A Gene Mutation Affecting Risk of Myocardial Infarction

INTRODUCTION: Multiple frequent genetic variants were shown to affect myocardial infarction (MI) risk. Genetic causes for familial clustering of MI are less clear. We aimed to identify and characterize the molecular underpinnings of premature MI in a family with 9 affected individuals. METHODS AND RESULTS: Employing cosegregation analysis and exome sequencing we identified a mutation in the phosphodiesterase 5A ( PDE5A ) gene in all affected individuals (LOD score 3.16). It is located in an alternative promoter site of PDE5A and leads to a premature stop codon in one of the PDE5A isoforms (p.Lys7Ter). PDE5A encodes for three isoforms catalyzing cGMP, a second messenger mediating vasodilation and platelet passivation. Effects of the stop codon were investigated by western blot analysis after in vitro mutagenesis. Overexpression in HEK cells did not reveal a loss of transcript but the expression of a N-terminally truncated protein. Deeper analyses of translation initiation by deletion of possible transcription starts via in vitro mutagenesis uncovered a protein lacking 91 amino acids compared to the full-length isoform. Activity of the truncated PDE5A was measured using PDEGlo (Promega). Moreover, the effect of the variant on the alternative promoter site was analyzed by luciferase assays. Therefore, a 600 bp fragment containing either the mutated or WT allele was cloned into a pGL4.10 vector (Promega). Overexpression in HEK cells showed 40% increase of promoter activity with the mutated allele (p&lt;0.05). Similar results could be shown in other cell lines. CONCLUSION: We identified a mutation in the PDE5A gene associated with premature MI. While a gain of function of PDE5A makes sense from a pathophysiological stance, the particular variant seemed to result in a premature stop codon. However, we could demonstrate that the variant might lead to increased promoter activity and that the presumable stop codon does not result into a loss of transcript but rather a truncated, potentially more active PDE5A isoform. Along with our homology modeling results and x-ray crystallographic and biochemical studies of Wang et al. (2010), these data support the idea of overexpression of a truncated though functional PDE5A in mutation carriers, possibly resulting in a gain function. </jats:p

Hidden Mutations in CdLS - Limitations of Sanger Sequencing in Molecular Diagnostics

International audienceCornelia de Lange syndrome (CdLS) is a well characterized developmental disorder. The genetic cause of CdLS is a mutation in one of five associated genes (NIPBL, SMC1A, SMC3, RAD21 and HDAC8) accounting for about 70 % of cases. To improve our current molecular diagnostic and to analyze some of CdLS candidate genes we developed and established a gene panel approach. Because recent data indicate a high frequency of mosaic NIPBL mutations that were not detected by conventional sequencing approaches of blood DNA, we started to collected buccal mucosa samples of our patients that were negative for mutations in the known CdLS genes. Here we report the identification of three mosaic NIPBL mutations by our high-coverage gene panel sequencing approach that were undetected by classical Sanger sequencing analysis of buccal mucosa DNA. All mutations were confirmed by the use of highly sensitive SNaPshot fragment analysis using DNA from buccal mucosa, urine and fibroblast samples. In blood samples we could not detect the respective mutation. Finally, in fibroblast samples from all three patients, Sanger sequencing could identify all the mutations. Thus, our study highlights the need for highly sensitive technologies in molecular diagnostic of CdLS to improve genetic diagnosis and counseling of patients and their families. This article is protected by copyright. All rights reserved