Particularidades genéticas y bioquímicas de la hipercolesterolemia familiar en el suroeste de la Península Ibérica / Genetics and biochemistry of familial hypercholesterolemia in Southwest of the Iberian Peninsula

Hasta el momento, la mayor parte de los casos de hipercolesterolemia familiar (60-80%) se atribuyen a variantes patogénicas en el gen LDLR. Solo un 1-5% de los casos se produce por variantes en el gen APOB y un 0-3% por variantes en el gen PCSK9. Existen gran variedad en mutaciones patogénicas conocidas del gen LDLR mientras que, para las que afectan al gen APOB, la de mayor incidencia es p.Arg3527Gln, descrita predominantemente en poblaciones de Centroeuropa y América del Norte. En la Península Ibérica el gen predominante afectado es el del receptor de LDL, similar al resto del mundo, siendo la afectación del gen APOB descrita en individuos del noroeste y anecdótica en el resto del territorio. Analizamos genéticamente la población asistida en el primer año de una consulta de lípidos del suroeste de España con puntuación≥6 puntos de las clínicas de lípidos holandesas y describimos los hallazgos genéticos, bioquímicos y clínicos. Los primeros hallazgos muestran indicios de una posible mayor prevalencia de pacientes con mutación en el gen APOB respecto a otros territorios. Encontramos hechos históricos que darían una posible explicación a este hecho, apoyando así dicha presunción.(AU)

So far, most cases of hypercholesterolaemia (60-80%) are attributed to pathogenic variants in the LDLR gene. Only 1-5% of cases are caused by variants in the APOB gene, and 0-3% by variants in the PCSK9 gene. There is a large variety in known pathogenic mutations of the LDLR gene, while for those affecting the APOB gene, the highest incidence is p.Arg3527Gln, described predominantly in Central European and North American populations. In the Iberian Peninsula the predominant gene affected is that of the LDL receptor, similar to the rest of the world, with the involvement of the APOB gene being described in individuals from the northwest, and anecdotal in the rest of the territory. A genetics analysis was performed on the population attending the first year of a lipid clinic in southwestern Spain with a 6-point score from the Dutch lipid clinics. The genetic, biochemical and clinical findings are described. The first findings show indications of a possible higher prevalence of patients with mutation in the APOB gene compared to other territories. Historical evidence is presented that could give a possible explanation to this, thus supporting the assumption.(AU)

Genetics and biochemistry of familial hypercholesterolemia in Southwest of the Iberian Peninsula. / Particularidades genéticas y bioquímicas de la hipercolesterolemia familiar en el suroeste de la Península Ibérica.

So far, most cases of hypercholesterolaemia (60-80%) are attributed to pathogenic variants in the LDLR gene. Only 1-5% of cases are caused by variants in the APOB gene, and 0-3% by variants in the PCSK9 gene. There is a large variety in known pathogenic mutations of the LDLR gene, while for those affecting the APOB gene, the highest incidence is p.Arg3527Gln, described predominantly in Central European and North American populations. In the Iberian Peninsula the predominant gene affected is that of the LDL receptor, similar to the rest of the world, with the involvement of the APOB gene being described in individuals from the northwest, and anecdotal in the rest of the territory. A genetics analysis was performed on the population attending the first year of a lipid clinic in southwestern Spain with a 6-point score from the Dutch lipid clinics. The genetic, biochemical and clinical findings are described. The first findings show indications of a possible higher prevalence of patients with mutation in the APOB gene compared to other territories. Historical evidence is presented that could give a possible explanation to this, thus supporting the assumption.

Effect of Evodia on Lipid Metabolism in Hyperlipidemia Mice / 中国实验方剂学杂志

Objective::To study the effect of evodia on lipid metabolism and low-density lipoprotein-receptor(LDL-R) mRNA expression in hyperlipidemia mice. Method::Kunming mice (n=80) were randomly divided into normal control group (n=20) and model group (n=60). Serum lipids of the model group were measured after 3 weeks.After successful modeling, the mice can be randomly divided into 5 groups (with 10 in each group): model group (equivalent normal saline), positive control group (simvastatin, 5 mg·kg-1·d-1), drug group (evodia of 5.25, 10.5, 21 mg·kg- 1·d- 1). The mice were given drugs for 3 weeks.Htoxylin-eosin(HE) staining was used to observe the liver cell structure and the change of aortic arch atherosclerosis in the mice.The enzyme linked immunosorbent assay kit was used to test the contents of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and total serum adiponectin (ADPN) in serum of the mice.The expression of LDL-R mRNA in liver of each group was detected by reverse transcription-polymerase chain reaction (RT-PCR). Result::Liver HE staining showed hepatocyte swelling with steatosis in the model group, and alleviated liver steatosis in high-dose, medium-dose evodia and simvastatin groups.HE staining showed damages on the aortict arch wall in the model group, with obvious intima thickening and inflammatory cell infiltration.The intima was thickened obviously in the low-dose group, and the structure of aortic vessel wall was clear in the high-dose group.Compared with the normal group, TC, TG and HDL-C levels in serum of the model group were increased, while HDL-C level was decreased (P<0.01). Serum TC and TG levels of mice in the medium and high-dose groups decreased, whereas LDL-C and HDLl-C levels increased in low, medium and high-dose groups (P<0.05, P<0.01). Compared with the normal group, the adiponectin level in the model group was decreased, while the serum adiponectin levels in medium and high-dose groups were significantly increased (P<0.01). The LDL-R mRNA expression in the liver of mice in the model group was significantly reduced compared with the normal group (P<0.01). The LDL-R mRNA expression in medium and high-dose evodia groups was significantly increased compared with the model group (P<0.01). Conclusion::Evodia can improve the tendency of hepatic lesions and aortic atherosclerosis in hyperlipidemia mice, which may be related to the regulation of adiponectin level, the reduction of lipid content in mice and the up-regulation of LDL-R mRNA expression in mice liver.

[PCSK9: Structure and function. PCSK9 and low-density lipoprotein receptor. Mutations and their effects]. / PCSK9: estructura y función. PCSK9 y receptor de lipoproteínas de baja densidad. Mutaciones y cambios derivados de estas.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low-density lipoprotein receptor (LDLr) and then targets it for lysosomal degradation in cells, thus preventing LDLr from recycling back to the hepatocyte surface, with a consequent decrease in LDLr density and clearance of LDL-cholesterol (LDLc). There have been reports of both gain-of-function mutations in the PCSK9 gene that cause a marked increase in LDLc conentrations and loss-of-function mutations, which lead to modest reductions in LDLc and low rates of coronary heart disease. The PCSK9 gene has become a promising therapeutic target to reduce blood cholesterol levels. This review discusses the most interesting recent data on PCSK9 regulation and its molecular function in cholesterol homeostasis.

LDL-R promoting activity of peptides derived from human PCSK9 catalytic domain (153-421): design, synthesis and biochemical evaluation.

BACKGROUND: High level of Low Density Lipoprotein-Cholesterol (LDL-C) in circulation in the blood is associated with an elevated risk of cardiovascular disease (CVD) and stroke. Currently the statin drugs which inhibit the enzyme HMG-CoA reductase responsible for cholesterol synthesis in the liver are very effective in lowering LDL-cholesterol. However these drugs are often associated with serious side effects particularly for ∼10-12% of cases. Therefore there is a need to develop non-statin based cholesterol reducing agents. Recently it was revealed that the secreted Proprotein Convertase Subtilisin Kexin 9 (PCSK9) binds with LDL-receptor (LDL-R) causing its degradation in the lysosome with the result of LDL-C accumulating in the blood. Thus PCSK9 has become an alternative target for development of non-statin cholesterol reducing agents. It is established that the catalytic domain of PCSK9 (aa153-421) and the EGF-A domain of LDL-R (aa314-355) are involved in the above bind leading to the reduction of LDL-R level and accumulation of LDL-C. OBJECTIVE: The major goal of this study is to identify peptide/s from the catalytic domain of hPCSK9 that can block the binding of hPCSK9 and LDL-R and therefore can reduce LDL-R degradation leading to the clearance of LDL-C from the plasma. RESULTS: Using 51 synthetic linear peptides (P1-P51) of 15aa long with 10 amino acids overlapping sequences spanning the entire catalytic segment of hPCSK9 (aa153-421), we identified two domains of hPCSK9 namely (aa323-358) and (aa365-384) that exhibited strong binding affinity towards synthetic EGF-A peptide. The results were based on mass spectrometry, fluorescence spectroscopy and native gel electrophoresis. Thus peptides containing the above segments in part (P35-P39 and P42-P47) exhibited LDL-R promoting activity when added exogenously to culture medium of growing human hepatic cells like HepG2 and HuH7. The effects were particularly significant with peptides P36, P37, P46 and P47. Interestingly, the first two peptides are present within the disulphide loop Cys(323)-Cys(358) and contain the key gain of function mutation D(374)/Y site while the last two peptides contain another disulphide bridge loop Cys(375)-Cys(378) and the second most potent gain of function mutation R(357)/H. Further studies revealed that S-S bridged cyclic loop peptide hPCSK9(365-384) exhibited the highest (∼3.5-fold) LDL-R promoting activity in both HepG2 and HuH7 when applied at 5 µM concentration level. This effect is completely abrogated when one of the Cys residues is substituted by Ala thereby preventing any S-S bond formation. This suggested its critical role in the bioactivity. It is proposed that LDL-R promoting activity of this and other selected PCSK9 catalytic peptides such as P36, P37, P46 and P47 are most likely mediated via intervention of PCSK9:LDL-R complex formation. Our findings may find useful application in future development of small molecule PCSK9 inhibitors for intervention of hypercholesterolemia and associated cardiovascular disease.

Single step PCR for the identification of Low Density Lipoprotein Receptor (LDL-R) gene mutations.

BACKGROUND AND OBJECTIVE: This study was conducted to determine the common mutation of low density lipoprotein receptor in patients with familial hypercholesterolemia (FH) in our population and identify the different point mutation in the LDL-receptor gene. The main aim of this study was to reduce the cost of PCR without extracting DNA and do the diagnosis at single step. METHODS: This study was carried out in the period of one year, from 2009- 2011. All the patients selected for this study were from Dr. Ziauddin Hospital, National Institute of Cardiovascular Diseases, and Dr. Rubina Ghani's Pathological & Molecular Laboratories. While collecting the blood sample, the patients were in overnight fasting condition. The clinical and biochemical analysis was performed on hyperlipidemic patients (n=120) to determine the frequency of familial hypercholesterolemia in our population. After lipid profile the patients were selected and direct multiplex PCR (Polymerase chain reaction) was performed from whole blood collected in a single tube using forward and reverse primers of exons 3, 4, 9 and 14 of without extracting DNA. RESULTS: Genomic DNA was extracted from blood samples as well as direct whole ETDA blood of healthy control group and hypercholesterolemia patients to detect mutations in exons 3, 4, 9, and 14 of the LDLR gene, with modification in the technique by using type-specific primers. These results for exon 4 mutation were confirmed by DNA sequencing. CONCLUSION: Screening method based on PCR by using Kappa direct PCR could be a faster and cheaper method with least contamination for screening a large number of FH patients for mutation of LDLR gene.

Angiotensin Ⅱ induces lipid accumulation in human renal proximal tubular epithelial cells via the disruption of low density lipoprotein receptor pathway / 中华肾脏病杂志

Objective To investigate the effects of angiotensin Ⅱ (Ang Ⅱ) stimulating on cholesterol influx in human renal proximal tubular epithelial cells (HK-2) and the relation to low-density lipoprotein receptor (LDLr) pathway.Methods HK-2 cells were cultured and divided into the control group (incubated with serum-free medium) and Ang Ⅱ group (treated by 10-7 mol/L of Ang Ⅱ for 24 hours).The effects of Ang Ⅱ on lipid accumulation were examined by Oil red O staining and a quantitative assay of intracellular cholesterol.The expression of LDLr,sterol regulatory elementbinding protein (SREBP) cleavage activating protein (SCAP) and SREBP-2 mRNA and protein were examined by real-time PCR and Western blotting.The cotranslocation of SCAP-SREBP-2 from endoplasmic retieulum to Golgi in HK-2 cells was examined by immunofluorescent staining under confocal microscopy.Results Ang Ⅱ treatment increased intracellular lipid accumulation in HK-2 cells,which was associated with increased mRNA and protein expression of LDLr,SCAP,and SREBP-2 in HK-2 cells induced by Ang Ⅱ.Furthermore,results from confocal microscopy observation demonstrated that Ang Ⅱ increased the translocation of SCAP/SREBP-2 complex from endoplasmic reticulum to Golgi,thereby up-regulating LDLr gene transcription.Conclusion Ang Ⅱ disrupts LDLr feed-back regulation to increase cholesterol uptake and induce intracellular lipid accumulation.