Novel PCSK9 (Proprotein Convertase Subtilisin Kexin Type 9) Variants in Patients With Familial Hypercholesterolemia From Cape Town.

OBJECTIVE: Familial hypercholesterolemia (FH) is characterized by elevated low-density lipoprotein-cholesterol and markedly increased cardiovascular risk. In patients with a genetic diagnosis, low-density lipoprotein receptor (LDLR) mutations account for >90% of cases, apolipoprotein B (APOB) mutations for ≈5% of cases, while proprotein convertase subtilisin kexin type 9 (PCSK9) gain of function mutations are rare (<1% of cases). We aimed to evaluate the functional impact of several novel PCSK9 variants in a cohort of patients with FH by genetic cascade screening and in vitro functionality assays. Approach and Results: Patients with clinically diagnosed FH underwent genetic analysis of LDLR, and if negative, sequential testing of APOB and PCSK9. We analyzed cosegregation of hypercholesterolemia with novel PCSK9 variants. Gain of function status was determined by in silico analyses and validated by in vitro functionality assays. Among 1055 persons with clinical FH, we identified nonsynonymous PCSK9 variants in 27 (2.6%) patients and 7 of these carried one of the 4 previously reported gain of function variants. In the remaining 20 patients with FH, we identified 7 novel PCSK9 variants. The G516V variant (c.1547G>T) was found in 5 index patients and cascade screening identified 15 additional carriers. Low-density lipoprotein-cholesterol levels were higher in these 15 carriers compared with the 27 noncarriers (236±73 versus 124±35 mg/dL; P<0.001). In vitro studies demonstrated the pathogenicity of the G516V variant. CONCLUSIONS: In our study, 1.14% of cases with clinical FH were clearly attributable to pathogenic variants in PCSK9. Pathogenicity is established beyond doubt for the G516V variant.

Wild antelope skeletal muscle antioxidant enzyme activities do not correlate with muscle fibre type or oxidative metabolism.

Wild antelope are some of the fastest land animals in the world, presenting with high oxidative and glycolytic skeletal muscle metabolism. However, no study has investigated their muscle antioxidant capacity, and may assist in understanding their physical ability and certain pathophysiological manifestations, such as capture myopathy. Therefore, the primary aim of this study was to determine the antioxidant activities superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR), as well as five key regulatory enzymes that serve as markers of glycolysis (phosphofructokinase (PFK) and lactate dehydrogenase (LDH)), the tricarboxylic acid cycle (citrate synthase (CS)), ß-oxidation (3-hydroxyacetyl CoA dehydrogenase (3HAD)) and the phosphagen pathway (creatine kinase (CK)), in the Vastus lateralis muscle of six southern African wild antelope species (mountain reedbuck, springbok, blesbok, fallow deer, black wildebeest and kudu). Four different muscle groups from laboratory rats served as reference values for the enzyme activities. SOD, CS and LDH activities were the highest in the wild antelope, whereas CK appeared highest in rat fast twitch muscles. Between the wild antelope species, differences exist for SOD, CAT, PFK, CK and LDH, but not for CS, 3HAD and GR. CAT and GR correlated positively only with type I fibres. No correlations could be found between muscle fibre type and the oxidative enzymes, CS and 3HAD, from the wild animals, concurring with previous studies on porcine and rats. However, wild antelope and rat muscle CK and SOD strongly correlated, hinting towards an antioxidant role for CK.

A novel mitochondrial DNA variant in MT-ND6: m.14430A>C p.(Trp82Gly) identified in a patient with Leigh syndrome and complex I deficiency.

Leigh syndrome is a severe progressive mitochondrial disorder mainly affecting children under the age of 5 years. It is caused by pathogenic variants in any one of more than 75 known genes in the nuclear or mitochondrial genomes. A 19-week-old male infant presented with lactic acidosis and encephalopathy following a 2-week history of irritability, neuroregression and poor weight gain. He was hypotonic with pathological reflexes, impaired vision, and nystagmus. Brain MRI showed extensive bilateral symmetrical T2 hyperintense lesions in basal ganglia, thalami, and brainstem. Metabolic workup showed elevated serum alanine, and heavy lactic aciduria with increased ketones, fumarate, malate, and alpha-ketoglutarate as well as reduced succinate on urine organic acid analysis. Lactic acidemia persisted, with only a marginally elevated lactate:pyruvate ratio (16.46, ref. 0-10). He demised at age 7 months due to respiratory failure. Exome sequencing followed by virtual gene panel analysis for pyruvate metabolism and mitochondrial defects could not identify any nuclear cause for Leigh syndrome. Mitochondrial DNA (mtDNA) genome sequencing revealed 88% heteroplasmy for a novel variant, NC_012920.1(MT-ND6):m.14430A>C p.(Trp82Gly), in blood DNA. This variant was absent from the unaffected mother's blood, fibroblast, and urine DNA, and detected at a level of 5% in her muscle DNA. Mitochondrial respiratory chain analysis revealed markedly reduced mitochondrial complex I activity in patient fibroblasts (34% of parent and control cells), and reduced NADH-linked respirometry (less than half of parental and control cells), while complex II driven respirometry remained intact. The combined clinical, genetic, and biochemical findings suggest that the novel MT-ND6 variant is the likely cause of Leigh syndrome in this patient. The mitochondrial ND6 protein is a subunit of complex I. An interesting finding was the absence of a significantly elevated lactate:pyruvate ratio in the presence of severe lactatemia, which directed initial diagnostic efforts towards excluding a pyruvate metabolism defect. This case highlights the value of a multidisciplinary approach and complete genetic workup to diagnosing mitochondrial disorders in South African patients.

Composition and distribution of lipoproteins after evolocumab in familial dysbetalipoproteinemia: A randomized controlled trial.

BACKGROUND: Proprotein convertase subtilisin kexin type 9 (PCSK9) monoclonal antibodies (mAbs) reduce fasting and post fat load cholesterol in non-HDL and intermediate density lipoprotein (IDL) in familial dysbetalipoproteinemia (FD). However, the effect of PCSK9 mAbs on the distribution and composition of atherogenic lipoproteins in patients with FD is unknown. OBJECTIVE: To evaluate the effect of the PCSK9 mAb evolocumab added to standard lipid-lowering therapy in patients with FD on fasting and post fat load lipoprotein distribution and composition. METHODS: Randomized placebo-controlled double-blind crossover trial comparing evolocumab (140 mg subcutaneous every 2 weeks) with placebo during two 12-week treatment periods. Patients received an oral fat load at the start and end of each treatment period. Apolipoproteins (apo) were measured with ultracentrifugation, gradient gel electrophoresis, retinyl palmitate and SDS-PAGE. RESULTS: PCSK9 mAbs significantly reduced particle number of all atherogenic lipoproteins, with a stronger effect on smaller lipoproteins than on larger lipoproteins (e.g. IDL-apoB 49%, 95%confidence interval (CI) 41-59 and very low-density lipoprotein (VLDL)-apoB 33%, 95%CI 16-50). Furthermore, PCSK9 mAbs lowered cholesterol more than triglyceride (TG) in VLDL, IDL and low-density lipoprotein (LDL) (e.g. VLDL-C 48%, 95%CI 29-63%; and VLDL-TG 20%, 95%CI 6.3-41%). PCSK9 mAbs did not affect the post fat load response of chylomicrons. CONCLUSION: PCSK9 mAbs added to standard lipid-lowering therapy in FD patients significantly reduced lipoprotein particle number, in particular the smaller and more cholesterol-rich lipoproteins (i.e. IDL and LDL). PCSK9 mAbs did not affect chylomicron metabolism. It seems likely that the observed effects are achieved by increased hepatic lipoprotein clearance, but the specific working mechanism of PCSK9 mAbs in FD patients remains to be elucidated.

Low-density lipoprotein cholesterol and non-high-density lipoprotein cholesterol measurement in Familial Dysbetalipoproteinemia.

AIM: To compare LDL-C concentrations using the Friedewald formula, the Martin-Hopkins formula, a direct assay and polyacrylamide gradient gel electrophoresis (PGGE) to the reference standard density gradient ultracentrifugation in patients with Familial Dysbetalipoproteinemia (FD) patients. We also compared non-HDL-cholesterol concentrations by two methods. METHODS: For this study data from 28 patients with genetically confirmed FD from the placebo arm of the EVOLVE-FD trial were used. Four different methods for determining LDL-C were compared with ultracentrifugation. Non-HDL-C was measured with standard assays and compared to ultracentrifugation. Correlation coefficients and Bland-Altman plots were used to compare the methods. RESULTS: Mean age of the 28 FD patients was 62 ± 9 years, 43 % were female and 93 % had an ɛ2ɛ2 genotype. LDL-C determined by Friedewald (R2 = 0.62, p <0.01), Martin-Hopkins (R2 = 0.50, p = 0.01) and the direct assay (R2 = 0.41, p = 0.03) correlated with density gradient ultracentrifugation. However, Bland-Altman plots showed considerable over- or underestimation by the four methods compared to ultracentrifugation. Non-HDL-C showed good correlation and agreement. CONCLUSION: In patients with FD, all four methods investigated over- or underestimated LDL-C concentrations compared with ultracentrifugation. In contrast, standard non-HDL-C assays performed well, emphasizing the use of non-HDL-C in patients with FD.

Effect of evolocumab on fasting and post fat load lipids and lipoproteins in familial dysbetalipoproteinemia.

BACKGROUND: Familial dysbetalipoproteinemia (FD) is the second most common monogenic lipid disorder (prevalence 1 in 850-3500), characterized by postprandial remnant accumulation and associated with increased cardiovascular disease (CVD) risk. Many FD patients do not achieve non-HDL-C treatment goals, indicating the need for additional lipid-lowering treatment options. OBJECTIVES: To evaluate the effect of the PCSK9 monoclonal antibody evolocumab added to standard lipid-lowering therapy on fasting and post fat load lipids and lipoproteins in patients with FD. METHODS: A randomized placebo-controlled double-blind crossover trial comparing evolocumab (140 mg subcutaneous every 2 weeks) with placebo during two 12-week treatment periods. At the start and end of each treatment period patients received an oral fat load. The primary endpoint was the 8-hour post fat load non-HDL-C area under the curve (AUC). Secondary endpoints included fasting and post fat load lipids and lipoproteins. RESULTS: In total, 28 patients completed the study. Mean age was 62±9 years and 93% had an Ɛ2Ɛ2 genotype. Evolocumab reduced the 8-hour post fat load non-HDL-C AUC with 49% (95%CI 42-55) and apolipoprotein B (apoB) AUC with 47% (95%CI 41-53). Other fasting and absolute post fat load lipids and lipoproteins including triglycerides and remnant-cholesterol were also significantly reduced by evolocumab. However, evolocumab did not have significant effects on the rise above fasting levels that occurred after consumption of the oral fat load. CONCLUSIONS: Evolocumab added to standard lipid-lowering therapy significantly reduced fasting and absolute post fat load concentrations of non-HDL-C, apoB and other atherogenic lipids and lipoproteins in FD patients. The clinically significant decrease in lipids and lipoproteins can be expected to translate into a reduction in CVD risk in these high-risk patients.

Hyperlipidemic myeloma, a rare form of acquired dysbetalipoproteinemia, in an HIV seropositive African female.

Dysbetalipoproteinemia (DBL) is an uncommon condition characterized by a mixed hyperlipidemia due to accumulation of remnant lipoproteins and is highly atherogenic. Typically, DBL is an autosomal recessive condition requiring an additional metabolic stress with reduced apolipoprotein E (apoE) function. However, DBL is also described in patients with multiple myeloma without the characteristic apoE2/E2 mutation seen in familial DBL. Although the underlying pathogenesis in these cases is not fully characterized, it is thought to occur due to interference with apoE function by antibodies produced from clonal plasma cells. Such cases are referred to as hyperlipidemic myeloma (HLM) and have rarely been described in the literature. To our knowledge there is no prior description of HLM in HIV positive patients in Africa. We describe a case of HLM in an African woman with underlying HIV infection who presented with phenotypic and biochemical features of DBL that responded poorly to lipid lowering therapy.

Melatonin protects against uric acid-induced mitochondrial dysfunction, oxidative stress, and triglyceride accumulation in C2C12 myotubes.

Excess uric acid has been shown to induce oxidative stress, triglyceride accumulation, and mitochondrial dysfunction in the liver and is an independent predictor of type-2 diabetes. Skeletal muscle plays a dominant role in type 2 diabetes and presents a large surface area to plasma uric acid. However, the effects of uric acid on skeletal muscle are underinvestigated. Our aim was therefore to characterize the effects of excessive uric acid on oxidative stress, triglyceride content, and mitochondrial function in skeletal muscle C2C12 myotubes and assess how these are modulated by the antioxidant molecule melatonin. Differentiated C2C12 myotubes were exposed to 750 µM uric acid or uric acid + 10 nM melatonin for 72 h. Compared with control, uric acid increased triglyceride content by ~237%, oxidative stress by 32%, and antioxidant capacity by 135%. Uric acid also reduced endogenous ROUTINE respiration, complex II-linked oxidative phosphorylation, and electron transfer system capacities. Melatonin counteracted the effects of uric acid without further altering antioxidant capacity. Our data demonstrate that excess uric acid has adverse effects on skeletal muscle similar to those previously reported in hepatocytes and suggest that melatonin at a low physiological concentration of 10 nM may be a possible therapy against some adverse effects of excess uric acid.NEW & NOTEWORTHY Few studies have investigated the effects of uric acid on skeletal muscle. This study shows that hyperuricemia induces mitochondrial dysfunction and triglyceride accumulation in skeletal muscle. The findings may explain why hyperuricemia is an independent predictor of diabetes.

Effects of rooibos (Aspalathus linearis) on oxidative stress and biochemical parameters in adults at risk for cardiovascular disease.

ETHNOPHARMACOLOGICAL RELEVANCE: In South Africa, the plant Aspalathus linearis (Brum.f) Dahlg. (Fabaceae) is traditionally used as a "tea" referred to as rooibos or redbush. This plant has been listed as a medicinal plant based mostly on anecdotal evidence. AIMS OF THE STUDY: Despite a long history of traditional use in South Africa, very little scientific data are available from controlled clinical trials confirming its popular use. The aim of the present study was to investigate the effect of rooibos on biochemical and oxidative stress parameters in adults at risk for cardiovascular disease. MATERIALS AND METHODS: After a washout period of 2 weeks, 40 volunteers consumed six cups of fermented/traditional rooibos daily for 6 weeks, followed by a control period. Blood biochemical parameters indicative of antioxidant activity and content (total polyphenols), lipid peroxidation (conjugated dienes - CDs, thiobarbituric acid reactive substances - TBARS), redox status (total glutathione - tGSH, ratio of reduced to oxidized glutathione - GSH:GSSG), lipid profile (total cholesterol, low density lipoprotein - LDL and high density lipoprotein - HDL cholesterol and triacylglycerol levels) and liver and kidney function were measured at the end of each study period. RESULTS: Plasma antioxidant capacity was not altered, but plasma total polyphenol levels increased significantly after rooibos consumption compared with the control levels (from 79.8±16.9 mg/L to 89.8±14.1 mg/L). Significant decreases in plasma markers of lipid peroxidation were found after rooibos consumption, as reported by levels of CDs (167.3±29.5 nmol/mL vs. 108.8±20.1 nmol/mL) and TBARS (1.9±0.6 µmol/L vs. 0.9±0.3 µmol/L). Reduced glutathione (797±238 µmol/L vs. 1082±140 µmol/L) and the GSH:GSSG ratio (41±14 vs. 76±17) were both significantly increased after consumption of rooibos. The lipid profiles showed that rooibos consumption, compared with the control values, significantly decreased serum LDL-cholesterol (4.6±1.3 mmol/L vs. 3.9±0.7 mmol/L) and triacylglycerols (1.7±0.8 mmol/L vs. 1.2±0.7 mmol/L), while HDL-cholesterol (0.9±0.1 mmol/L vs. 1.2±0.2 mmol/L) was significantly increased. CONCLUSION: Confirming its popular use, consumption of fermented, traditional rooibos significantly improved the lipid profile as well as redox status, both relevant to heart disease, in adults at risk for developing cardiovascular disease.