Trond P. Leren

Risk Factors Related to Carotid Intima-Media Thickness and Plaque in Children With Familial Hypercholesterolemia and Control Subjects

To assess the relationship between risk factors for cardiovascular disease and early atherosclerotic changes in the carotid artery, we measured carotid intima-media thickness by B-mode ultrasonography in 61 boys and 29 girls 10 to 19 years old with familial hypercholesterolemia (FH) and 30 control subjects matched for age and sex. All were nonsmokers, and all the FH adolescents had a known mutation in the LDL receptor gene. Mean intima-media thickness in the far wall of the carotid bulb was greater (P = .03) in the FH group than in the control subjects: 0.54 mm (95% confidence interval [CI], 0.52 to 0.56) versus 0.50 mm (95% CI, 0.47 to 0.52). In the entire group, mean and maximum intima-media thicknesses in the carotid bulb were positively associated with levels of apolipoprotein B and fibrinogen after control for pubertal stage (r = .19 to .24; P < .05), as was male sex. Plasma total homocysteine was similar in the FH and control groups and was associated with mean and maximum intima-media thicknesses in the far wall of the common carotid artery and carotid bulb after control for pubertal stage (r = .22 to .28; P < .05). With the exception of the relation between plasma fibrinogen level and mean carotid bulb intima-media thickness, these associations were essentially unchanged in stepwise multiple linear regression analyses, allowing for the entry of BMI and level of HDL cholesterol into the analysis. Carotid artery plaque was present in 10% of the children with FH versus none of the control subjects. Children with plaque had a higher mean cholesterol-years score than children without plaque. These findings suggest that the classic lipid and hemostatic risk factors as well as plasma total homocysteine are associated with markers of early carotid atherosclerosis from the second decade of life. B-mode ultrasonography may prove to be a useful tool in risk stratification of children with FH.

Missense Mutations in the PCSK9 Gene Are Associated With Hypocholesterolemia and Possibly Increased Response to Statin Therapy

Objective—The proprotein convertase subtilisin/kexin type 9 (PCSK9) gene encodes a proprotein convertase that causes degradation of cell surface low-density lipoprotein receptors (LDLRs). Mutations in the PCSK9 gene that disrupt the normal function of PCSK9 could therefore result in increased number of LDLRs and hypocholesterolemia. Also, the cholesterol-lowering effect of statins could be increased in subjects carrying mutations in the PCSK9 gene. Methods and Results—We have screened 38 unrelated hypocholesterolemic subjects as well as 25 unrelated familial hypercholesterolemia (FH) heterozygotes who responded particularly well to statin therapy for mutations in the 12 exons of the PCSK9 gene by DNA sequencing. Six of the 38 (15.8%) hypocholesterolemic subjects were heterozygous for 1 of the 3 mutations R46L, G106R, or R237W in the PCSK9 gene. In the group of 25 FH heterozygotes who responded particularly well to statin therapy, 3 (8.8%) were heterozygous for mutations R46L or N157K in the PCSK9 gene. None of 441 hypercholesterolemic subjects without mutations in the LDLR gene or in the apolipoprotein B-100 gene possessed any of the 4 mutations. Conclusion—The 4 missense mutations R46L, G106R, N157K, and R237W are associated with hypocholesterolemia and possibly increased response to statin therapy.

Berberine decreases PCSK9 expression in HepG2 cells

BACKGROUND Proprotein convertase subtilisin/kexin type 9 (PCSK9) post-transcriptionally downregulates the low-density lipoprotein receptor (LDLR) by binding to the receptors epidermal growth factor repeat A on the cell surface and shuttling the LDLR to the lysosomes for degradation. Mutations in the PCSK9 gene have been shown to cause either hypo- or hypercholesterolemia. Here we investigated the effect of berberine, a natural plant extract, on PCSK9 expression in HepG2 cells. RESULTS Berberine decreases PCSK9 mRNA and protein levels in a time- and dose-dependent manner. This was not due to increased degradation of PCSK9 mRNA but most likely due to a decreased transcription of the PCSK9 gene. We also show that a combination of berberine and mevastatin increases LDLR mRNA and protein levels, while suppressing the increase in PCSK9 mRNA levels caused by mevastatin alone. CONCLUSION Berberine may be a useful supplement to statin treatment due to its effects on PCSK9 mRNA and protein levels.

Prospective, population-based long QT molecular autopsy study of postmortem negative sudden death in 1 to 40 year olds

BACKGROUND Retrospective investigation of sudden unexplained death in the young (SUDY) reveals that a high proportion is due to inherited heart disease. OBJECTIVE The purpose of this study was to ascertain the diagnostic value of postmortem long QT (LQT) genetic analysis in a prospective study of SUDY victims 1-40 years old. METHODS Denaturing high-performance liquid chromatography or direct sequencing of LQT genes 1, 2, 3, 5, and 6 was performed, in a National New Zealand protocol, in SUDY victims aged 1-40 years. RESULTS Over 26 months (2006-2008), DNA was stored at autopsy from 52 victims of sudden unexpected death. Further testing revealed a diagnosis in 19 cases (poisoning 4, dilated cardiomyopathy 3, myocarditis 3, other 9). The remaining 33 cases underwent genetic testing (age at death 18 months-40 years, median 25 years). Eighteen (55%) died during sleep or at rest, and 7 (21%) died during light activity. Rare missense variants in LQT genes were found in 5 (15%) cases (confidence interval 3%-27%): T96R in KCNQ1 (11-year-old male), P968L in KCNH2 (32-year-old female), P2006A in SCN5A (34-year-old female), and R67H and R98W in KCNE1 (17- and 38-year-old females, respectively). Evidence of pathogenicity was provided by in vitro evidence (T96R), family phenotype-genotype co-segregation (R98W, P2006A), and/or previous reports (R67H, P968L, P2006A, R98W). Family cardiac investigation was possible in 23 (70%) families and revealed probable cause of death for 5 (15%) other victims (confidence interval 3%-27%). CONCLUSION Most community SUDY occurs at rest or during light activity. A diagnostic rate of 15% supports the transition of LQT genetic autopsy, combined with family investigation, into routine medical practice.

Left ventricular mechanical dispersion by tissue Doppler imaging: a novel approach for identifying high-risk individuals with long QT syndrome

Aims The aim of this study was to investigate whether prolonged and dispersed myocardial contraction duration assessed by tissue Doppler imaging (TDI) may serve as risk markers for cardiac events (documented arrhythmia, syncope, and cardiac arrest) in patients with long QT syndrome (LQTS). Methods and results Seventy-three patients with genetically confirmed LQTS (nine double- and 33 single-mutation carriers with previous cardiac events and 31 single-mutation carriers without events) were studied. Myocardial contraction duration was prolonged in each group of LQTS patients compared with 20 healthy controls (P < 0.001). Contraction duration was longer in single-mutation carriers with previous cardiac events compared with those without (0.46 ± 0.06 vs. 0.40 ± 0.06 s, P = 0.001). Prolonged contraction duration could better identify cardiac events compared with corrected QT (QTc) interval in single-mutation carriers [area under curve by receiver-operating characteristic analysis 0.77 [95% confidence interval (95% CI) 0.65–0.89] vs. 0.66 (95% CI 0.52–0.79)]. Dispersion of contraction was more pronounced in single-mutation carriers with cardiac events compared with those without (0.048 ± 0.018 vs. 0.031 ± 0.019 s, P = 0.001). Conclusion Dispersion of myocardial contraction assessed by TDI was increased in LQTS patients. Prolonged contraction duration was superior to QTc for risk assessment. These new methods can easily be implemented in clinical routine and may improve clinical management of LQTS patients.

Right ventricular mechanical dispersion is related to malignant arrhythmias: a study of patients with arrhythmogenic right ventricular cardiomyopathy and subclinical right ventricular dysfunction.

AIMS We evaluated if right ventricular (RV) mechanical dispersion by strain was related to ventricular arrhythmias (VT/VF) in patients with arrhythmogenic right ventricular cardiomyopathy (ARVC) and if mechanical dispersion was increased in so far asymptomatic mutation carriers. METHODS AND RESULTS We included 69 patients, 42 had symptomatic ARVC and 27 were mutation positive asymptomatic family members. Forty healthy individuals served as controls. Myocardial strain was assessed in 6 RV and 16 left ventricular (LV) segments. Contraction duration (CD) in 6 RV and 16 LV segments were measured as the time from onset R on electrocardiogram to maximum myocardial shortening in each segment. The standard deviation of CD was defined as mechanical dispersion. Mechanical dispersion was more pronounced in ARVC patients with arrhythmias compared with asymptomatic mutation carriers and healthy individuals in RV [52(41,63) vs. 35(23,47) vs. 13(9,19)ms, P < 0.001]. Mechanical dispersion was more pronounced in asymptomatic mutation carriers compared with healthy individuals (P < 0.001). Right ventricular mechanical dispersion predicted VT/VF in a multivariate logistic regression analysis [odds ratio (OR), 1.66 (95% confidence interval (CI) 1.06-2.58), P < 0.03]. Right ventricular and LV function by strain were reduced in symptomatic ARVC patients and correlated significantly (R = 0.81, P < 0.001). Right ventricular and LV strain were reduced in asymptomatic mutation carriers compared with healthy individuals (P < 0.001). CONCLUSION Right ventricular mechanical dispersion was pronounced in patients with ARVC with VT/VF. Right ventricular mechanical dispersion was present in asymptomatic mutation carriers and may be helpful in risk stratification. Right ventricular and LV function correlated in ARVC patients implying that ARVC is a biventricular disease.

Transmural Differences in Myocardial Contraction in Long-QT Syndrome Mechanical Consequences of Ion Channel Dysfunction

Background— Long-QT syndrome (LQTS) is characterized by prolonged myocardial action potential duration. The longest action potential duration is reported in the endomyocardium and midmyocardium. Prolonged action potential duration in LQTS may cause prolonged cardiac contraction, which can be assessed by strain echocardiography. We hypothesized that myocardial contraction is most prolonged in subendocardial myofibers in LQTS patients and that inhomogeneous transmural contraction is related to the risk of spontaneous arrhythmia. Methods and Results— We included 101 genotyped LQTS mutation carriers and 35 healthy individuals. A history of cardiac arrhythmias was present in 48 mutations carriers, and 53 were asymptomatic. Myocardial contraction duration was assessed by strain echocardiography as time from the ECG Q wave to peak strain in 16 LV segments. Strain was assessed along the longitudinal axis, predominantly representing subendocardial fibers, and along the circumferential axis, representing midmyocardial fibers. Mean contraction duration was longer in LQTS mutation carriers compared with healthy individuals (445±45 versus 390±40 milliseconds; P<0.001) and longer in symptomatic compared with asymptomatic LQTS mutation carriers (460±40 versus 425±45 milliseconds; P<0.001). Contraction duration by longitudinal strain was longer than by circumferential strain in symptomatic LQTS patients (460±45 versus 445±45 milliseconds; P=0.008) but not in asymptomatic patients and healthy individuals, indicating transmural mechanical dispersion. This time difference was present in a majority of LV segments and was most evident in patients with LQT2 and the Jervell and Lange-Nielsen syndrome. Conclusion— Contraction duration in symptomatic LQTS mutation carriers was longer in the subendocardium than in the midmyocardium, indicating transmural mechanical dispersion, which was not present in asymptomatic and healthy individuals.Background— Long-QT syndrome (LQTS) is characterized by prolonged myocardial action potential duration. The longest action potential duration is reported in the endomyocardium and midmyocardium. Prolonged action potential duration in LQTS may cause prolonged cardiac contraction, which can be assessed by strain echocardiography. We hypothesized that myocardial contraction is most prolonged in subendocardial myofibers in LQTS patients and that inhomogeneous transmural contraction is related to the risk of spontaneous arrhythmia. Methods and Results— We included 101 genotyped LQTS mutation carriers and 35 healthy individuals. A history of cardiac arrhythmias was present in 48 mutations carriers, and 53 were asymptomatic. Myocardial contraction duration was assessed by strain echocardiography as time from the ECG Q wave to peak strain in 16 LV segments. Strain was assessed along the longitudinal axis, predominantly representing subendocardial fibers, and along the circumferential axis, representing midmyocardial fibers. Mean contraction duration was longer in LQTS mutation carriers compared with healthy individuals (445±45 versus 390±40 milliseconds; P <0.001) and longer in symptomatic compared with asymptomatic LQTS mutation carriers (460±40 versus 425±45 milliseconds; P <0.001). Contraction duration by longitudinal strain was longer than by circumferential strain in symptomatic LQTS patients (460±45 versus 445±45 milliseconds; P =0.008) but not in asymptomatic patients and healthy individuals, indicating transmural mechanical dispersion. This time difference was present in a majority of LV segments and was most evident in patients with LQT2 and the Jervell and Lange-Nielsen syndrome. Conclusion— Contraction duration in symptomatic LQTS mutation carriers was longer in the subendocardium than in the midmyocardium, indicating transmural mechanical dispersion, which was not present in asymptomatic and healthy individuals. # Clinical Perspective {#article-title-34}

Effects of lovastatin alone and in combination with cholestyramine on serum lipids and apolipoproteins in heterozygotes for familial hypercholesterolemia

We have studied the effect of lovastatin, an inhibitor of the rate-limiting enzyme in cholesterol biosynthesis (3-hydroxy-3-methylglutaryl coenzyme A reductase), alone and in combination with the bile acid sequestrant cholestyramine on lipid parameters in 30 heterozygous patients with familial hypercholesterolemia (FH) during a 20-week open trial. Lovastatin 40 mg bid (twice daily) decreased significantly total serum cholesterol, low density lipoprotein (LDL)-cholesterol, triglycerides and apolipoprotein B by 36%, 45%, 29% and 11%, respectively, while high density lipoprotein (HDL)-cholesterol and apolipoprotein A-I were increased significantly by 16% and 37%, respectively. These data are consistent with a reduction in both the number of LDL particles and in their cholesterol content. Addition of cholestyramine 4 g bid caused a significant further decrease in total serum cholesterol and LDL-cholesterol to a total of 43% and 61%, respectively. The addition of 4 g bid or 8 g bid of cholestyramine caused only minor changes in the other lipid parameters. No effect was found by these drugs on Lp(a) lipoprotein level. We conclude that lovastatin alone or in combination with a small dose of cholestyramine normalizes the lipid profile in most FH heterozygotes.

Description of a large family with autosomal dominant hypercholesterolemia associated with the APOE p.Leu167del mutation

Apolipoprotein (apo) E mutants are associated with type III hyperlipoproteinemia characterized by high cholesterol and triglycerides levels. Autosomal dominant hypercholesterolemia (ADH), due to the mutations in the LDLR, APOB, or PCSK9 genes, is characterized by an isolated elevation of cholesterol due to the high levels of low‐density lipoproteins (LDLs). We now report an exceptionally large family including 14 members with ADH. Through genome‐wide mapping, analysis of regional/functional candidate genes, and whole exome sequencing, we identified a mutation in the APOE gene, c.500_502delTCC/p.Leu167del, previously reported associated with sea‐blue histiocytosis and familial combined hyperlipidemia. We confirmed the involvement of the APOE p.Leu167del in ADH, with (1) a predicted destabilization of an alpha‐helix in the binding domain, (2) a decreased apo E level in LDLs, and (3) a decreased catabolism of LDLs. Our results show that mutations in the APOE gene can be associated with bona fide ADH.

Retention of Mutant Low Density Lipoprotein Receptor in Endoplasmic Reticulum (ER) Leads to ER Stress

Familial hypercholesterolemia is an autosomal dominant disease caused by mutations in the gene encoding the low density lipoprotein receptor (LDLR). More than 50% of these mutations lead to receptor proteins that are completely or partly retained in the endoplasmic reticulum (ER). The mechanisms involved in the intracellular processing and retention of mutant LDLR are poorly understood. In the present study we show that the G544V mutant LDLR associates with the chaperones Grp78, Grp94, ERp72, and calnexin in the ER of transfected Chinese hamster ovary cells. Retention of the mutant LDLR was shown to cause ER stress and activation of the unfolded protein response. We observed a marked increase in the activity of two ER stress sensors, IRE1 and PERK. These results show that retention of mutant LDLR in ER induces cellular responses, which might be important for the clinical outcome of familial hypercholesterolemia.