Knut Erik Berge

Overexpression of ABCG5 and ABCG8 promotes biliary cholesterol secretion and reduces fractional absorption of dietary cholesterol

Two ATP-binding cassette (ABC) transporters, ABCG5 and ABCG8, have been proposed to limit sterol absorption and to promote biliary sterol excretion in humans. To test this hypothesis, a P1 clone containing the human ABCG5 and ABCG8 genes was used to generate transgenic mice. The transgenes were expressed primarily in the liver and small intestine, mirroring the expression pattern of the endogenous genes. Transgene expression only modestly affected plasma and liver cholesterol levels but profoundly altered cholesterol transport. The fractional absorption of dietary cholesterol was reduced by about 50%, and biliary cholesterol levels were increased more than fivefold. Fecal neutral sterol excretion was increased three- to sixfold and hepatic cholesterol synthesis increased two- to fourfold in the transgenic mice. No significant changes in the pool size, composition, and fecal excretion of bile acids were observed in the transgenic mice. Transgene expression attenuated the increase in hepatic cholesterol content induced by consumption of a high cholesterol diet. These results demonstrate that increased expression of ABCG5 and ABCG8 selectively drives biliary neutral sterol secretion and reduces intestinal cholesterol absorption, leading to a selective increase in neutral sterol excretion and a compensatory increase in cholesterol synthesis.

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.

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}

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.

Characterization of novel mutations in the catalytic domain of the PCSK9 gene

Objectives.  To expand our understanding of the structure and function of proprotein convertase subtilisin/kexin type 9 (PCSK9) by studying how naturally occurring mutations in PCSK9 disrupt the function of PCSK9.

Diagnosis of familial hypercholesterolemia in general practice using clinical diagnostic criteria or genetic testing as part of cascade genetic screening.

Background: Too few familial hypercholesterolemia (FH) patients are diagnosed. The most cost-effective strategy to diagnose FH is to examine first-degree relatives of already diagnosed patients. This is referred to as cascade genetic screening. Methods and Results: One thousand eight hundred and five first-degree relatives of index patients with molecularly defined FH consented to cascade genetic screening by the use of molecular genetic testing. Of these, 44.8% were mutation carriers and 55.2% were noncarriers. Only 44.2% of the mutation carriers were on lipid-lowering drugs at the time of genetic testing. Of these, only 9.4% had a value for total serum cholesterol below 5 mM. Among adult mutation carriers who were not on lipid-lowering treatment at the time of genetic testing, reductions in total serum cholesterol and low-density lipoprotein cholesterol of 18.4% (p < 0.0001) and 25.3% (p < 0.0001), respectively, were observed 6 months after genetic testing. It is assumed that this improvement in the lipid profile is due to a definite diagnosis obtained by molecular genetic testing. By using the results of genetic testing as the gold standard for diagnosis of FH, data from a questionnaire filled out by the relatives showed that the use of clinical criteria to diagnose FH in general practice had a sensitivity of 46.2% and a specificity of 88.0%. Conclusions: The use of clinical diagnostic criteria to diagnose FH in general practice identifies only approximately 50% of FH patients. Molecular genetic testing as part of cascade genetic screening is an efficient tool to diagnose patients, leading to significant improvement in the lipid profile. It should therefore be implemented in clinical medicine.

The prevalence of mutations in KCNQ1, KCNH2, and SCN5A in an unselected national cohort of young sudden unexplained death cases.

Introduction: Sudden unexplained death account for one‐third of all sudden natural deaths in the young (1–35 years). Hitherto, the prevalence of genopositive cases has primarily been based on deceased persons referred for postmortem genetic testing. These deaths potentially may represent the worst of cases, thus possibly overestimating the prevalence of potentially disease causing mutations in the 3 major long‐QT syndrome (LQTS) genes in the general population. We therefore wanted to investigate the prevalence of mutations in an unselected population of sudden unexplained deaths in a nationwide setting.

High prevalence of exercise-induced arrhythmias in catecholaminergic polymorphic ventricular tachycardia mutation-positive family members diagnosed by cascade genetic screening

AIM Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac disease predisposing to life-threatening arrhythmias. We aimed to determine the prevalence of arrhythmias and efficacy of beta-blocker treatment in mutation-positive family members diagnosed by cascade genetic screening. METHODS AND RESULTS Relatives of six unrelated CPVT patients were tested for the relevant mutation in the ryanodine receptor-2 gene. Mutation carriers underwent an exercise test at inclusion time and 3 months after the initiation of beta-blocker therapy in the highest tolerable dose. The occurrence of ventricular premature beats, couplets, and non-sustained ventricular arrhythmias (nsVT) were recorded in addition to the heart rate at which they occurred. Thirty family members were mutation carriers and were followed for 22 (13-288) months. Previous undiagnosed CPVT-related symptoms were reported by eight subjects. Exercise test induced ventricular arrhythmias in 23 of the 30 mutation carriers. On beta-blocker treatment, exercise-induced arrhythmias occurred at a lower heart rate (117 +/- 17 vs. 135 +/- 34 beats/min, P = 0.02) but at similar workload (P = 0.78). Beta-blocker treatment suppressed the occurrence of exercise-induced nsVT in three of the four patients, while less severe arrhythmias were unchanged. One patient died during follow-up. CONCLUSION Exercise test revealed a high prevalence of arrhythmias in CPVT mutation carriers diagnosed by cascade genetic screening. beta-Blocker therapy appeared to suppress the most severe exercise-induced arrhythmias, while less severe arrhythmias occurred at a lower heart rate.

Investigations on the evolutionary conservation of PCSK9 reveal a functionally important protrusion

Proprotein convertase subtilisin/kexin type 9 (PCSK9) interferes with the recycling of low‐density lipoprotein (LDL) receptor (LDLR). This leads to LDLR degradation and reduced cellular uptake of plasma LDL. Naturally occurring human PCSK9 loss‐of‐function mutations are associated with low levels of plasma LDL cholesterol and a reduced risk of coronary heart disease. PCSK9 gain‐of‐function mutations result in lower LDL clearance and increased risk of atherosclerosis. The exact mechanism by which PCSK9 disrupts the normal recycling of LDLR remains to be determined. In this study, we have assembled homologs of human PCSK9 from 20 vertebrates, a cephalochordate and mollusks in order to search for conserved regions of PCSK9 that may be important for the PCSK9‐mediated degradation of LDLR. We found a large, conserved protrusion on the surface of the PCSK9 catalytic domain and have performed site‐directed mutagenesis experiments for 13 residues on this protrusion. A cluster of residues that is important for the degradation of LDLR by PCSK9 was identified. Another cluster of residues, at the opposite end of the conserved protrusion, appears to be involved in the physical interaction with a putative inhibitor of PCSK9. This study identifies the residues, sequence segments and surface patches of PCSK9 that are under strong purifying selection and provides important information for future studies of PCSK9 mutants and for investigations on the function of this regulator of cholesterol homeostasis.