Low-density Lipoprotein Receptor Adaptor Protein Research Articles

The diagnostic value of genetic testing in familial hypercholesterolemia in patients with premature myocardial infarction

Objective: To evaluate the diagnostic value of gene testing in familial hypercholesterolemia (FH) in patients with premature myocardial infarction(PMI). Methods: This study was a single center cross-sectional study. A retrospective analysis was made on PMI patients who visited the People's Hospital of Peking University from May 1, 2015 to March 31, 2017. Clinical data of patients was collected and gene testing of FH related genes low density lipoprotein receptor (LDLR), proprotein convertase subtilisin/kexin type 9 (PCSK9), apolipoprotein B(APOB) and low density lipoprotein receptor adaptor protein 1(LDLRAP1) was carried out. Clinical diagnosis of FH patients was performed using Simon Broome criteria, DLCN criteria, and FH Chinese expert consensus. Results: There were 188 males (83.6%) among 225 PMI patients, and the age of the first myocardial infarction was (46.6±7.2) years old. Ten patients carried FH pathogenic or possibly pathogenic mutations (4.4%). Compared with Simon Broome standard, DLCN standard and FH Chinese expert consensus, gene testing increased the diagnostic rate of FH by 53.3%, 33.3% and 42.1% respectively. Conclusion: Gene testing is helpful to improve the diagnosis of FH, and it is important to start the standard treatment of FH as early as possible in patients with premature myocardial infarction.

Familial Hypercholesterolemia: A Literature Review of the Pathophysiology and Current and Novel Treatments.

Familial hypercholesterolemia (FH) is a genetically transmitted disorder. It shows an autosomal dominant mode of inheritance. It is a metabolic disorder. Mutation in chromosome 19 leads to this disorder. Chromosome 19 codes data for low-density lipoprotein (LDL) receptor (LDLR). LDLR either reduces increased LDL levels from the circulation or maintains a normal level of LDL. It precipitates the risk of cardiovascular disease at an early age. Characteristic features of FH are elevated levels of LDL in the blood because of sudden changes in LDLR, which causes a decrease in the clearance of LDL from the blood. Plaque gets deposited in the lumen of the arteries, called atherosclerosis, which occurs at an early young age. If both genes are affected then it is homozygous FH (HoFH); such a case is very rare. When a single gene is affected then that condition is known as heterozygous FH (HeFH). HoFH comes up with more severe cardiac disease than HeFH at an early age. The major cause of FH is a mutation in the LDLR gene while other causes include mutation in various genes like apolipoprotein B (apo B), proprotein convertase subtilisin/kexin type 9 (PCSK9), LDLR adaptor protein 1 (LDLRAP 1). In order to prevent cardiovascular crises due to FH, it must be diagnosed early and treated effectively. With increasing research and advances in medical sciences, many kinds of current and novel therapies are emerging that aim to reduce the level of LDL in blood.

LDLR and PCSK9 3´UTR variants and their putative effects on microRNA molecular interactions in familial hypercholesterolemia: a computational approach.

Familial hypercholesterolemia (FH) is caused by pathogenic variants in low-density lipoprotein (LDL) receptor (LDLR) or its associated genes, including apolipoprotein B (APOB), proprotein convertase subtilisin/kexin type 9 (PCSK9), and LDLR adaptor protein 1 (LDLRAP1). However, approximately 40% of the FH patients clinically diagnosed (based on FH phenotypes) may not carry a causal variant in a FH-related gene. Variants located at 3' untranslated region (UTR) of FH-related genes could elucidate mechanisms involved in FH pathogenesis. This study used a computational approach to assess the effects of 3'UTR variants in FH-related genes on miRNAs molecular interactions and to explore the association of these variants with molecular diagnosis of FH. Exons and regulatory regions of FH-related genes were sequenced in 83 FH patients using an exon-target gene sequencing strategy. In silico prediction tools were used to study the effects of 3´UTR variants on interactions between miRNAs and target mRNAs. Pathogenic variants in FH-related genes (molecular diagnosis) were detected in 44.6% FH patients. Among 59 3'UTR variants identified, LDLR rs5742911 and PCSK9 rs17111557 were associated with molecular diagnosis of FH, whereas LDLR rs7258146 and rs7254521 and LDLRAP1 rs397860393 had an opposite effect (p < 0.05). 3´UTR variants in LDLR (rs5742911, rs7258146, rs7254521) and PCSK9 (rs17111557) disrupt interactions with several miRNAs, and more stable bindings were found with LDLR (miR-4435, miR-509-3 and miR-502) and PCSK9 (miR-4796). LDLR and PCSK9 3´UTR variants disturb miRNA:mRNA interactions that could affect gene expression and are potentially associated with molecular diagnosis of FH.

Reducing Premature Coronary Artery Disease in Malaysia by Early Identification of Familial Hypercholesterolemia Using the Familial Hypercholesterolemia Case Ascertainment Tool (FAMCAT): Protocol for a Mixed Methods Evaluation Study.

Familial hypercholesterolemia (FH) is predominantly caused by mutations in the 4 FH candidate genes (FHCGs), namely, low-density lipoprotein receptor (LDLR), apolipoprotein B-100 (APOB-100), proprotein convertase subtilisin/kexin type 9 (PCSK9), and the LDL receptor adaptor protein 1 (LDLRAP1). It is characterized by elevated low-density lipoprotein cholesterol (LDL-c) levels leading to premature coronary artery disease. FH can be clinically diagnosed using established clinical criteria, namely, Simon Broome (SB) and Dutch Lipid Clinic Criteria (DLCC), and can be identified using the Familial Hypercholesterolemia Case Ascertainment Tool (FAMCAT), a primary care screening tool. This study aims to (1) compare the detection rate of genetically confirmed FH and diagnostic accuracy between the FAMCAT, SB, and DLCC in the Malaysian primary care setting; (2) identify the genetic mutation profiles, including novel variants, in individuals with suspected FH in primary care; (3) explore the experience, concern, and expectation of individuals with suspected FH who have undergone genetic testing in primary care; and (4) evaluate the clinical utility of a web-based FH Identification Tool that includes the FAMCAT, SB, and DLCC in the Malaysian primary care setting. This is a mixed methods evaluation study conducted in 11 Ministry of Health primary care clinics located at the central administrative region of Malaysia. In Work stream 1, the diagnostic accuracy study design is used to compare the detection rate and diagnostic accuracy of the FAMCAT, SB, and DLCC against molecular diagnosis as the gold standard. In Work stream 2, the targeted next-generation sequencing of the 4 FHCGs is used to identify the genetic mutation profiles among individuals with suspected FH. In Work stream 3a, a qualitative semistructured interview methodology is used to explore the experience, concern, and expectation of individuals with suspected FH who have undergone genetic testing. Lastly, in Work stream 3b, a qualitative real-time observation of primary care physicians using the "think-aloud" methodology is applied to evaluate the clinical utility of a web-based FH Identification Tool. The recruitment for Work stream 1, and blood sampling and genetic analysis for Work stream 2 were completed in February 2023. Data collection for Work stream 3 was completed in March 2023. Data analysis for Work streams 1, 2, 3a, and 3b is projected to be completed by June 2023, with the results of this study anticipated to be published by December 2023. This study will provide evidence on which clinical diagnostic criterion is the best to detect FH in the Malaysian primary care setting. The full spectrum of genetic mutations in the FHCGs including novel pathogenic variants will be identified. Patients' perspectives while undergoing genetic testing and the primary care physicians experience in utilizing the web-based tool will be established. These findings will have tremendous impact on the management of patients with FH in primary care and subsequently reduce their risk of premature coronary artery disease. DERR1-10.2196/47911.

How Genetic Variants in Children with Familial Hypercholesterolemia Not Only Guide Detection, but Also Treatment

Familial hypercholesterolemia (FH) is a hereditary disorder that causes severely elevated low-density lipoprotein (LDL-C) levels, which leads to an increased risk for premature cardiovascular disease. A variety of genetic variants can cause FH, namely variants in the genes for the LDL receptor (LDLR), apolipoprotein B (APOB), proprotein convertase subtilisin/kexin type 9 (PCSK9), and/or LDL-receptor adaptor protein 1 (LDLRAP1). Variants can exist in a heterozygous form (HeFH) or the more severe homozygous form (HoFH). If affected individuals are diagnosed early (through screening), they benefit tremendously from early initiation of lipid-lowering therapy, such as statins, and cardiovascular imaging to detect possible atherosclerosis. Over the last years, due to intensive research on the genetic basis of LDL-C metabolism, novel, promising therapies have been developed to reduce LDL-C levels and subsequently reduce cardiovascular risk. Results from studies on therapies focused on inhibiting PCSK9, a protein responsible for degradation of the LDLR, are impressive. As the effect of PCSK9 inhibitors (PCSK9-i) is dependent of residual LDLR activity, this medication is less potent in patients without functional LDLR (e.g., null/null variant). Novel therapies that are expected to become available in the near future focused on inhibition of another major regulatory protein in lipid metabolism (angiopoietin-like 3 (ANGPTL3)) might dramatically reduce the frequency of apheresis in children with HoFH, independently of their residual LDLR. At present, another independent risk factor for premature cardiovascular disease, elevated levels of lipoprotein(a) (Lp(a)), cannot be effectively treated with medication. Further understanding of the genetic basis of Lp(a) metabolism, however, offers a possibility for the development of novel therapies.

Gene and cell therapy approaches for familial hypercholesterolemia: An update.

Familial hypercholesterolemia (FH) is a common autosomal codominant hereditary illness marked by the heightened risk of early atherosclerotic cardiovascular disease and high blood levels of low-density lipoprotein cholesterol (LDL-C). FH patients can have homozygous or heterozygous variants. This condition has been linked to variations in the genes for the LDL receptor (LDLR), apolipoprotein B, proprotein convertase subtilisin/Kexin 9 (PCSK9), and LDLR adaptor protein 1. Drugs such as statins, ezetimibe, and PCSK9 inhibitors are currently widely available, allowing for the theoretical normalization of plasma LDL-C levels mostly in patients with heterozygous FH. However, homozygous FH patients usually have a poor response to traditional lipid-lowering therapy and may have a poor prognosis at a young age. LDL apheresis and novel pharmacological therapies such as microsomal transfer protein inhibitors or anti-angiopoietin-like protein 3 monoclonal antibodies are extremely expensive and unavailable in most regions of the world. Therefore, the unmet need persists for these patients. In this review, we discuss the numerous gene delivery, gene editing, and stem cell manipulation techniques used in this study to correct FH-causing LDLR gene variations in vitro, ex vivo, and in vivo. Finally, we looked at a variety of studies that corrected genetic defects that caused FH using the ground-breaking clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing technology.

Genetic Spectrum of Familial Hypercholesterolaemia in the Malaysian Community: Identification of Pathogenic Gene Variants Using Targeted Next-Generation Sequencing

Familial hypercholesterolaemia (FH) is caused by mutations in lipid metabolism genes, predominantly in low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), proprotein convertase subtilisin/kexin-type 9 (PCSK9) and LDL receptor adaptor protein 1 (LDLRAP1). The prevalence of genetically confirmed FH and the detection rate of pathogenic variants (PV) amongst clinically diagnosed patients is not well established. Targeted next-generation sequencing of LDLR, APOB, PCSK9 and LDLRAP1 was performed on 372 clinically diagnosed Malaysian FH subjects. Out of 361 variants identified, 40 of them were PV (18 = LDLR, 15 = APOB, 5 = PCSK9 and 2 = LDLRAP1). The majority of the PV were LDLR and APOB, where the frequency of both PV were almost similar. About 39% of clinically diagnosed FH have PV in PCSK9 alone and two novel variants of PCSK9 were identified in this study, which have not been described in Malaysia and globally. The prevalence of genetically confirmed potential FH in the community was 1:427, with a detection rate of PV at 0.2% (12/5130). About one-fourth of clinically diagnosed FH in the Malaysian community can be genetically confirmed. The detection rate of genetic confirmation is similar between potential and possible FH groups, suggesting a need for genetic confirmation in index cases from both groups. Clinical and genetic confirmation of FH index cases in the community may enhance the early detection of affected family members through family cascade screening.

Deletion of LDLRAP1 Induces Atherosclerotic Plaque Formation, Insulin Resistance, and Dysregulated Insulin Response in Adipose Tissue

Hypercholesterolemia, vascular inflammation, obesity, and insulin resistance often overlap and exacerbate each other. Mutations in low density lipoprotein receptor adaptor protein‐1 (LDLRAP1) lead to LDL receptor malfunction and are associated with the Autosomal Recessive Hypercholesterolemia (ARH) disorder in humans; however, direct causality on atherogenesis in a defined pre‐clinical model has not been reported. The objective of this study was to test the hypothesis that deletion of LDLRAP1 will lead to hypercholesteremia and atherosclerosis. LDLRAP1‐/‐ mice fed a high‐fat western diet (HFD) had significantly increased plasma cholesterol and triglyceride concentrations (p&lt;0.001), accompanied with significantly increased plaque burden compared with wild‐type controls. Unexpectedly, LDLRAP1‐/‐ mice gained significantly more weight compared with controls (p&lt;0.001), and even on a chow diet, LDLRAP1‐/‐ mice are insulin resistant, and calorimetric studies suggests an altered metabolic profile. We determined that LDLRAP1 is highly expressed in white adipose tissue (VAT), and LDLRAP1‐/‐ adipocytes are significantly larger (p&lt;0.01), and have reduced glucose uptake and AKT phosphorylation, but increased CD36 expression. VAT from LDLRAP1‐/‐ mice is hypoxic and has gene expression signatures of dysregulated lipid storage and energy homeostasis. These data are the first to indicate that lack of LDLRAP1 directly leads to atherosclerosis in mice and also plays an unanticipated metabolic regulatory role in adipose tissue. LDLRAP1 may link atherosclerosis and hypercholesterolemia with common co‐morbidities of obesity and insulin resistance.

Deletion of LDLRAP1 Induces Atherosclerotic Plaque Formation, Insulin Resistance, and Dysregulated Insulin Response in Adipose Tissue

Dyslipidemia, vascular inflammation, obesity, and insulin resistance often overlap and exacerbate each other. Mutations in low density lipoprotein receptor adaptor protein-1 (LDLRAP1) lead to LDLR malfunction and are associated with the autosomal recessive hypercholesterolemia disorder in humans. However, direct causality on atherogenesis in a defined preclinical model has not been reported. The objective of this study was to test the hypothesis that deletion of LDLRAP1 will lead to hypercholesteremia and atherosclerosis. LDLRAP1-/- mice fed a high-fat Western diet had significantly increased plasma cholesterol and triglyceride concentrations accompanied with significantly increased plaque burden compared with wild-type controls. Unexpectedly, LDLRAP1-/- mice gained significantly more weight compared with controls. Even on a chow diet, LDLRAP1-/- mice were insulin-resistant, and calorimetric studies suggested an altered metabolic profile. The study showed that LDLRAP1 is highly expressed in visceral adipose tissue, and LDLRAP1-/- adipocytes are significantly larger, have reduced glucose uptake and AKT phosphorylation, but have increased CD36 expression. Visceral adipose tissue from LDLRAP1-/- mice was hypoxic and had gene expression signatures of dysregulated lipid storage and energy homeostasis. These data are the first to indicate that lack of LDLRAP1 directly leads to atherosclerosis in mice and also plays an unanticipated metabolic regulatory role in adipose tissue. LDLRAP1 may link atherosclerosis and hypercholesterolemia with common comorbidities of obesity and insulin resistance.

Abstract 13123: Global Knockout of LDLRAP1 Regulates Atherosclerosis, Adipose Metabolism, and Insulin Resistance

Introduction: Atherosclerotic vascular syndromes account for 50% of all deaths in the United States, and are a considerable medical and socioeconomic problem contributing to mortality of several conditions, including myocardial infarction and stroke. It is accepted that atherosclerosis and symptoms of metabolic syndrome, such as obesity, insulin resistance, and hypercholesterolemia often coincide with and exacerbate one another, however the molecular events in common with these conditions are not yet fully understood. One event that may link these two disorders is lipid receptor trafficking and signaling of the LDL receptor (LDLR), which plays a key role in LDL uptake and regulation of plasma LDL concentration. Low-density lipoprotein receptor adaptor protein 1 (LDLRAP1) is a cytosolic adaptor protein which interacts with the cytoplasmic tail of the LDLR, mediating internalization of the LDL-LDLR complex and connecting it to the cell’s endocytic machinery. The purpose of this study was to characterize the effects of LDLRAP1 global knockout on atherogenesis and metabolic function in order to gain a better understanding of the molecular mechanisms that interconnect atherosclerosis and metabolic syndrome. Results: LDLRAP1 global knockout mice fed a high-fat western diet (HFD) for 16 weeks displayed increased atherosclerotic plaque burden (p&lt;0.0001), serum cholesterol (p&lt;0.0001) and triglyceride levels (p&lt;0.01), and expression of lipid receptors SR-A1 and CD36 (p&lt;0.05), and gained significantly more weight compared to controls (p&lt;0.0001). LDLRAP1 -/- adipose also displayed decreased glucose uptake (p&lt;0.01) and pAKT and AKT protein levels (p&lt;0.05). Both before and after a HFD, LDLRAP1 -/- mice were also significantly insulin resistant (p&lt;0.01) with concomitant dysregulated insulin signaling in adipose tissue, and displayed metabolic differences as quantified by metabolic cage analysis (p&lt;0.05). Conclusions: These data suggest that in addition to a function in cholesterol internalization, LDLRAP1 participates in regulating adipose metabolism and insulin sensitivity. This implicates LDLRAP1 as a key molecule and potential therapeutic target in treatment of diseases which constitute metabolic syndrome.

Familial Hypercholesterolemia: Global Burden and Approaches.

Familial hypercholesterolemia (FH) is the most common genetic metabolic disorder characterized by markedly elevated LDL-C levels from birth leading to atherosclerotic cardiovascular disease (ASCVD) and premature deaths. The purpose of this review is to share the current knowledge in the diagnosis, risk estimation, and management of patients with FH in the light of recent evidence and guideline recommendations. Recent registries underscored the prevalence of FH as 1/200-250 translating to an almost 1500 million subjects suffering from FH worldwide. However, only a minority of FH patients are identified early and effectively treated. In most cases, mutations in the LDL-receptor (LDLR) gene and to a lesser degree in the apolipoprotein B-100 (APOB), proprotein convertase subtilisin/kexin type 9 (PCSK9), and the LDL-receptor adaptor protein 1 (LDLRAP1) genes cause FH. Diagnostic scores such as Dutch Lipid Clinic Network criteria using clinical manifestations are helpful in identifying FH. Traditional risk factors and high lipoprotein(a) affect the course of the disease. Vascular ultrasound imaging and coronary calcium scoring are helpful for further risk estimation of these patients. Getting to LDL-C goals is possible with currently available treatments including statins, ezetimibe, and PCSK9 inhibitors, as well as lipoprotein apheresis, lomitapide, and mipomersen in more severe phenotypes. Additionally, novel agents bempedoic acid, inclisiran, and evinacumab expanded the treatment choices for some patients with FH. Early diagnosis and initiation of LDL-C lowering are still required to achieve the greatest reduction in ASCVD morbidity and mortality in patients with FH. FH is a common genetic disorder characterized by markedly elevated LDL-C levels from birth onward, resulting in significantly increased risk for ASCVD. Despite major advances in our understanding of the disease and effective therapies, FH is still underdiagnosed and undertreated. Early initiation of LDL-C lowering by increased awareness of FH among the healthcare professionals, patients, and the public is necessary to achieve meaningful reduction in ASCVD morbidity and mortality in these patients.

Comparison of The Two Lipoprotein Apheresis Methods in The Treatment of Familial Hypercholesterolemia

Objective: Familial hypercholesterolemia (FH) is an autosomal dominant inherited genetic disorder that causes a significant increase in low-density lipoprotein (LDL) cholesterol levels and leads to early coronary heart disease and cardiac mortality. Although this disease has a heterozygous (HeFH) and homozygous (HoFH) form, the incidence of HeFH is reported to be 1: 500, while HoFH is reported to be 1: 1 000 000. This disease is often caused by LDL receptor (most common), apolipoprotein B (Apo B), Proprotein Convertase Subtilisin/Kexin 9 (PCSK9), and LDL receptor adaptor protein (LDLRAP) gene mutations. Patients with FH do not respond well to lipid-lowering therapies such as a statin, and so lipoprotein apheresis is the treatment of choice. Material and Methods: We compared the results of lipoprotein apheresis in a 20-year-old female patient diagnosed with HoFH with two different methods [double-filtration plasmapheresis (DFPP) and dextran sulfate column (DSC) methods]. 40 lipoprotein apheresis procedures including 20 sessions of DFPP and 20 sessions of DSC were evaluated. Results: When the two methods were compared the changes in high-density lipoprotein, white blood cells, platelets, potassium, calcium, prothrombin time (INR) and activated partial thromboplastin time values were statistically significant. Conclusions: In conclusion, our study suggests that it would be more appropriate to choose the apheresis method according to the clinical and laboratory findings of the patient since it shows that two different methods have different effects on serum electrolyte values, hemostasis criteria and leukocyte and platelet counts.

Persistent hypercholesterolemia in child with homozygous autosomal recessive hypercholesterolemia: A decade of lipid management

Autosomal recessive hypercholesterolemia (ARH) is a rare form of genetic hypercholesterolemia caused by mutations in low density lipoprotein receptor adaptor protein 1 (LDLRAP1). The proband first presented with linear eruptive xanthomas over her ankles, knees and elbows, with low density lipoprotein cholesterol (LDL-C) of 16.0 mmol/L (618.7 mg/dL), at 2.5 years old. Next generation sequencing revealed a novel homozygous mutation in LDLRAP1 exon 5 (c.466delG).In the first year, drug regimens of either cholestyramine or simvastatin, reduced her LDL-C to 10.5 mmol/L (406 mg/dL) and 11.7 mmol/L (452.4 mg/dL), respectively. Combination simvastatin and ezetimibe was the mainstay of therapy from age 5 - 10 years. Her lowest achieved LDL-C was 6.3 mmol/L (243.6 mg/dL). Switching to atorvastatin did not lead to further reduction. Carotid intima-media thickness was 0.47 mm (> 97th percentile) and 0.32 mm (75 - 95th percentile) at ages 8 years and 11 years, respectively. Addition of monthly injections of evolocumab for 3 months, led to an increase in LDL-C, from 7.0 mmol/L (270.7 mg/dL) to a range of [(8.4 - 9.1) mmol/L or (324.8 - 351.9) mg/dL].In this report, a decade-long lipid management is described in a patient with ARH. Residual activity of LDLRAP1 is a likely determinant of her response. Clinical management remains sub-optimal and options for the paediatric population are limited. Novel classes of cholesterol-lowering medications are needed for this ultra-rare and severe hypercholesterolemia.

Cascade Screening for Familial Hypercholesterolemia in South Africa: The Wits FIND-FH Program.

Due to gene founder effects, familial hypercholesterolemia (FH) has a prevalence of ≈1:80 in populations of Afrikaner ancestry and is a major contributor to premature atherosclerotic cardiovascular disease in South Africans of Jewish and Indian descent. No systematic program exists to identify these families. Furthermore, information regarding FH prevalence in Black Africans is sparse. The Wits FIND-FH program was initiated in late 2016 to address these issues. Approach and Results: Based on index subjects with definite or probable FH, first-degree relatives were contacted, informed consent obtained, and targeted medical history, physical examination, and blood samples collected. In patients with likely FH using the Simon Broome criteria, DNA analysis for LDLR (low-density lipoprotein receptor), APOB (apolipoprotein B), PCSK9 (proprotein convertase subtilisin/kexin type 9), and LDLRAP1 (LDLR adaptor protein 1) variants was analyzed by next-generation sequencing. Of the initial 700 subjects screened of whom 295 (42%) were index cases, 479 (68.4%) were clinically diagnosed with probable or definite FH. Genetic analysis confirmed 285 of 479 (59.5%) as having variants consistent with FH. Three subjects met the clinical diagnosis for homozygous FH, but DNA analysis revealed a further 34 patients, including 4 Black African subjects, with ≥2 FH-causing variants. Using phenotype cascade screening, the Wits FIND-FH program has screened an average of 30 subjects monthly of whom 68% had a clinical diagnosis of FH with ≈60% genetically confirmed. The program is identifying a small but growing number of Black South Africans with FH. Interestingly, 37 subjects (7.7%) who underwent DNA testing were found to have ≥2 FH-causing variants.

Salvia miltiorrhiza-Derived Sal-miR-58 Induces Autophagy and Attenuates Inflammation in Vascular Smooth Muscle Cells

Autophagy is associated with the cytoprotection of physiological processes against inflammation and oxidative stress. Salvia miltiorrhiza possesses cardiovascular protective actions and has powerful anti-oxidative and anti-inflammatory effects; however, whether and how Salvia miltiorrhiza-derived microRNAs (miRNAs) protect vascular smooth muscle cells (VSMCs) by inducing autophagy across species are unknown. We first screened and identified Sal-miR-58 from Salvia miltiorrhiza as a natural autophagy inducer. Synthetic Sal-miR-58 suppresses chronic angiotensin II (Ang II) infusion-induced abdominal aortic aneurysm (AAA) formation in mice, as well as induces autophagy in VSMCs and attenuates the inflammatory response elicited by Ang II in vivo and in vitro. Mechanistically, Sal-miR-58 downregulates Krüppel-like factor 3 (KLF3) expression through direct binding to the 3′ UTR of KLF3, which in turn relieves KLF3 repression of E3 ubiquitin ligase neural precursor cell-expressed developmentally downregulated 4-like (NEDD4L) expression, whereas NEDD4L upregulation increases the ubiquitination and degradation of the platelet isoform of phosphofructokinase (PFKP), subsequently leading to a decrease in the activation of Akt/mammalian target of rapamycin (mTOR) signaling and facilitating VSMC autophagy induced by Sal-miR-58 in the context of chronic Ang II stimulation and aneurysm formation. Our results provide the first evidence that plant-derived Sal-miR-58 induces autophagy and attenuates inflammation in VSMCs through cross-species modulation of the KLF3/NEDD4L/PFKP regulatory pathway.

Molecular insights into the coding region mutations of low-density lipoprotein receptor adaptor protein 1 (LDLRAP1) linked to familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is a lipid disorder caused by pathogenic mutations in LDLRAP1 gene. The present study has aimed to deepen our understanding about the pathogenicity predictions of FH causative genetic mutations, as well as their relationship to phenotype changes in LDLRAP1 protein, by utilizing multidirectional computational analysis. FH linked LDLRAP1 mutations were mined from databases, and the prediction ability of several pathogenicity classifiers against these clinical variants, was assessed through different statistical measures. Furthermore, these mutations were 3D modelled in protein structures to assess their impact on protein phenotype changes. Our findings suggest that Polyphen-2, when compared with SIFT, M-CAP and CADD tools, can make better pathogenicity predictions for FH causative LDLRAP1 mutations. Through, 3D simulation and superimposition analysis of LDLRAP1 protein structures, it was found that missense mutations do not create any gross changes in the protein structure, although they could induce subtle structural changes at the level of amino acid residues. Near native molecular dynamic analysis revealed that missense mutations could induce variable degrees of fluctuation differences guiding the protein flexibility. Stability analysis showed that most missense mutations shifts the free energy equilibrium and hence they destabilize the protein. Molecular docking analysis demonstrates the molecular shifts in hydrogen and ionic bonds and Van der waals bonding properties, which further cause differences in the binding energy of LDLR-LDLRAP1 proteins. The diverse computational approaches used in the present study may provide a new dimension for exploring the structure-function relationship of the novel and deleterious LDLRAP1 mutations linked to FH.

Genetic associations between serum low LDL-cholesterol levels and variants in LDLR, APOB, PCSK9 and LDLRAP1 in African populations

Non-communicable diseases, including cardiovascular diseases (CVDs), are increasing in African populations. High serum low density lipoprotein cholesterol (LDL-cholesterol) levels are a known risk factor for CVDs in European populations, but the link remains poorly understood among Africans. This study investigated the associations between serum LDL-cholesterol levels and selected variants in the low density lipoprotein receptor (LDLR), apolipoprotein B (APOB), proprotein convertase subtilisin/kexin type 9 (PCSK9) and low density lipoprotein receptor adaptor protein 1 (LDLRAP1) genes in some selected African populations. Nineteen SNPs were selected from publicly available African whole genome sequence data based on functional prediction and allele frequency. SNPs were genotyped in 1000 participants from the AWI-Gen, study selected from the extremes of LDL-cholesterol level distribution (500 with LDL-cholesterol>3.5 mmol/L and 500 with LDL-cholesterol<1.1 mmol/L). The minor alleles at five of the six associated SNPs were significantly associated (P<0.05) with lower LDL-cholesterol levels: LDLRAP1 rs12071264 (OR 0.56, 95% CI: 0.39–0.75, P = 2.73x10-4) and rs35910270 (OR 0.78, 95% CI: 0.64–0.94, P = 0.008); APOB rs6752026 (OR 0. 55, 95% CI: 0.41–0.72, P = 2.82x10-5); LDLR: rs72568855 (OR 0.47, 95% CI: 0.27–0.82, P = 0.008); and PCSK9 rs45613943 (OR = 0.72, 95% CI: 0.58–0.88, P = 0.001). The minor allele of the sixth variant was associated with higher LDL-cholesterol levels: APOB rs679899 (OR 1.41, 95% CI: 1.06–1.86, P = 0.016). A replication analysis in the Africa America Diabetes Mellitus (AADM) study found the PCSK9 variant to be significantly associated with low LDL-cholesterol levels (Beta = -0.10). Since Africans generally have lower LDL-cholesterol levels, these LDL-cholesterol associated variants may be involved in adaptation due to unique gene-environment interactions. In conclusion, using a limited number of potentially functional variants in four genes, we identified significant associations with lower LDL-cholesterol levels in sub-Saharan Africans.

Genes Potentially Associated with Familial Hypercholesterolemia.

This review addresses the contribution of some genes to the phenotype of familial hypercholesterolemia. At present, it is known that the pathogenesis of this disease involves not only a pathological variant of low-density lipoprotein receptor and its ligands (apolipoprotein B, proprotein convertase subtilisin/kexin type 9 or low-density lipoprotein receptor adaptor protein 1), but also lipids, including sphingolipids, fatty acids, and sterols. The genetic cause of familial hypercholesterolemia is unknown in 20%–40% of the cases. The genes STAP1 (signal transducing adaptor family member 1), CYP7A1 (cytochrome P450 family 7 subfamily A member 1), LIPA (lipase A, lysosomal acid type), ABCG5 (ATP binding cassette subfamily G member 5), ABCG8 (ATP binding cassette subfamily G member 8), and PNPLA5 (patatin like phospholipase domain containing 5), which can cause aberrations of lipid metabolism, are being evaluated as new targets for the diagnosis and personalized management of familial hypercholesterolemia.

Molecular Characterization of Familial Hypercholesterolemia in a North American Cohort.

Familial hypercholesterolemia (FH) confers a very high risk of premature cardiovascular disease and is commonly caused by mutations in low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), or proprotein convertase subtilisin/kexin type 9 (PCSK9) and very rarely in LDLR adaptor protein 1 (LDLRAP1) genes. To determine the prevalence of pathogenic mutations in the LDLR, APOB, and PCSK9 in a cohort of subjects who met Simon Broome criteria for FH and compare the clinical characteristics of mutation-positive and mutation-negative subjects. Ninety-three men and 107 women aged 19 to 80 years from lipid clinics in the United States and Canada participated. Demographic and historical data were collected, physical examination performed, and serum lipids/lipoproteins analyzed. Targeted sequencing analyses of LDLR and PCSK9 coding regions and exon 26 of APOB were performed followed by detection of LDLR deletions and duplications. Disease-causing LDLR and APOB variants were identified in 114 and 6 subjects, respectively. Of the 58 LDLR variants, 8 were novel mutations. Compared with mutation-positive subjects, mutation-negative subjects were older (mean 49 years vs 57 years, respectively) and had a higher proportion of African Americans (1% vs 12.5%), higher prevalence of hypertension (21% vs 46%), and higher serum triglycerides (median 86 mg/dL vs 122 mg/dL) levels. LDLR mutations were the most common cause of heterozygous FH in this North American cohort. A strikingly high proportion of FH subjects (40%) lacked mutations in known culprit genes. Identification of underlying genetic and environmental factors in mutation-negative patients is important to further our understanding of the metabolic basis of FH and other forms of severe hypercholesterolemia.

Evaluation of the role of STAP1 in Familial Hypercholesterolemia

Familial hypercholesterolemia (FH) is characterised by elevated serum levels of low-density lipoprotein cholesterol (LDL-C) and a substantial risk for cardiovascular disease. The autosomal-dominant FH is mostly caused by mutations in LDLR (low density lipoprotein receptor), APOB (apolipoprotein B), and PCSK9 (proprotein convertase subtilisin/kexin). Recently, STAP1 has been suggested as a fourth causative gene. We analyzed STAP1 in 75 hypercholesterolemic patients from Berlin, Germany, who are negative for mutations in canonical FH genes. In 10 patients with negative family history, we additionally screened for disease causing variants in LDLRAP1 (low density lipoprotein receptor adaptor protein 1), associated with autosomal-recessive hypercholesterolemia. We identified one STAP1 variant predicted to be disease causing. To evaluate association of serum lipid levels and STAP1 carrier status, we analyzed 20 individuals from a population based cohort, the Cooperative Health Research in South Tyrol (CHRIS) study, carrying rare STAP1 variants. Out of the same cohort we randomly selected 100 non-carriers as control. In the Berlin FH cohort STAP1 variants were rare. In the CHRIS cohort, we obtained no statistically significant differences between carriers and non-carriers of STAP1 variants with respect to lipid traits. Until such an association has been verified in more individuals with genetic variants in STAP1, we cannot estimate whether STAP1 generally is a causative gene for FH.