Algorithm for detection and screening of familial hypercholesterolemia in Lithuanian population.

BACKGROUND: Familial hypercholesterolemia (FH) is one of the most common autosomal dominant diseases. FH causes a lifelong increase in low-density lipoprotein cholesterol (LDL-C) levels, which in turn leads to atherosclerotic cardiovascular disease. The incidence of FH is widely underestimated and undertreated, despite the availability and effectiveness of lipid-lowering therapy. Patients with FH have an increased cardiovascular risk; therefore, early diagnosis and treatment are vital. To address the burden of FH, several countries have implemented national FH screening programmes. The currently used method for FH detection in Lithuania is mainly based on opportunistic testing with subsequent cascade screening of index cases' first-degree relatives. METHODS: A total of 428 patients were included in this study. Patients with suspected FH are referred to a lipidology center for thorough evaluation. Patients who met the criteria for probable or definite FH according to the Dutch Lipid Clinic Network (DLCN) scoring system and/or had LDL-C > = 6.5 mmol/l were subjected to genetic testing. Laboratory and instrumental tests, vascular marker data of early atherosclerosis, and consultations by other specialists, such as radiologists and ophthalmologists, were also recorded. RESULTS: A total of 127/428 (30%) patients were genetically tested. FH-related mutations were found in 38.6% (n = 49/127) of the patients. Coronary artery disease (CAD) was diagnosed in 13% (n = 57/428) of the included patients, whereas premature CAD was found in 47/428 (11%) patients. CAD was diagnosed in 19% (n = 9/49) of patients with FH-related mutations, and this diagnosis was premature for all of them. CONCLUSIONS: Most patients in this study were classified as probable or possible FH without difference of age and sex. The median age of FH diagnosis was 47 years with significantly older females than males, which refers to the strong interface of this study with the LitHir programme. CAD and premature CAD were more common among patients with probable and definite FH, as well as those with an FH-causing mutation. The algorithm described in this study is the first attempt in Lithuania to implement a specific tool which allows to maximise FH detection rates, establish an accurate diagnosis of FH, excluding secondary causes of dyslipidaemia, and to select patients for cascade screening initiation more precisely.

Analysis of the Correlation between FH and PCSK9 and APOB Gene Mutations in Han and Mongolian Populations of the Hulunbuir.

BACKGROUND: The goal of the study was to provide an individual and precise genetic and molecular biological basis for the early prevention, diagnosis, and treatment of local FH by analyzing the risk factors for the development of FH in Han and Mongolian patients in the Hulunbuir, comparing the lipid levels of FH patients of the two ethnicities, and assessing differences in mutations to two genes between the two ethnic groups. METHODS: Twenty cases each of Han Chinese and Mongolian healthy controls and fifty patients who each met the inclusion criteria from November 2021 to December 2022 in five general hospitals in Hulunbuir were selected. Multifactor logistic analysis was used to analyze the risk factors associated with the development of FH. We used t-tests to analyze statistical differences in lipid levels between the groups, and Sanger sequencing to detect the dis-tribution of common mutation sites of PCSK9 and APOB in all study subjects. The mutation rates and differences between regions and ethnic groups were summarized and compared. RESULTS: 1) Gender, age, alcohol consumption, dietary status, and a family history of FH were risk factors associated with the development of FH. 2) TC, LDL-C, and APOB were significantly higher in Mongolian cases than Han cases (p < 0.05). sdLDL-C was not statistically different between the two ethnicities (p > 0.05). 3) We detected four (8%) heterozygous mutations at the PCSK9 gene E670G mutation site in the Han case group and a total of nine (18%) mutations at this site in the Mongolian cases, including one (2%) homozygous and eight (16%) heterozygous mutations. One case of a heterozygous mutation was detected in the Mongolian control group. We detected a total of ten (20%) mutations at the APOB gene rs1367117 mutation site in the Han case group, including eight (16%) heterozygous and two (4%) homozygous mutations, 11 cases (22%) of heterozygous mutations in the Mongolian case group, two cases of heterozygous mutations in the Han control group, and one case of a heterozygous mutation in the Mongolian control group. 4) The D374Y and S127R mutation sites of PCSK9 and the R3500Q mutation site of APOB were not detected in any of the study subjects. CONCLUSIONS: The mutation sites of the PCSK9 and APOB genes in FH patients in Hulunbuir are different from other regions, and the mutation rate is higher than in other regions. Therefore, we recommend that the mutation sites of the PCSK9 and APOB genes described herein be used as clinical detection indicators to assist the diagnosis of FH in this region.

Gallic Acid Can Promote Low-Density Lipoprotein Uptake in HepG2 Cells via Increasing Low-Density Lipoprotein Receptor Accumulation.

Gallic acid (GA) is a type of polyphenolic compound that can be found in a range of fruits, vegetables, and tea. Although it has been confirmed it improves non-alcoholic fatty liver disease (NAFLD), it is still unknown whether GA can improve the occurrence of NAFLD by increasing the low-density lipoprotein receptor (LDLR) accumulation and alleviating cholesterol metabolism disorders. Therefore, the present study explored the effect of GA on LDLR and its mechanism of action. The findings indicated that the increase in LDLR accumulation in HepG2 cells induced by GA was associated with the stimulation of the epidermal growth factor receptor-extracellular regulated protein kinase (EGFR-ERK1/2) signaling pathway. When the pathway was inhibited by EGFR mab cetuximab, it was observed that the activation of the EGFR-ERK1/2 signaling pathway induced by GA was also blocked. At the same time, the accumulation of LDLR protein and the uptake of LDL were also suppressed. Additionally, GA can also promote the accumulation of forkhead box O3 (FOXO3) and suppress the accumulation of hepatocyte nuclear factor-1α (HNF1α), leading to the inhibition of proprotein convertase subtilisin/kexin 9 (PCSK9) mRNA expression and protein accumulation. This ultimately results in increased LDLR protein accumulation and enhanced uptake of LDL in cells. In summary, the present study revealed the potential mechanism of GA's role in ameliorating NAFLD, with a view of providing a theoretical basis for the dietary supplementation of GA.

Association of lipid-lowering drug targets with risk of cutaneous melanoma: a mendelian randomization study.

BACKGROUND: Melanoma proliferation is partly attributed to dysregulated lipid metabolism. The effectiveness of lipid-lowering drugs in combating cutaneous melanoma (CM) is a subject of ongoing debate in both in vitro and clinical studies. METHOD: This study aims to evaluate the causal relationship between various lipid-lowering drug targets, namely 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR, targeted by statins), Proprotein convertase subtilisin/kexin type 9 (PCSK9, targeted by alirocumab and evolocumab), and Niemann-Pick C1-like 1 (NPC1L1, targeted by ezetimibe), and the outcomes of cutaneous melanoma. To mimic the effects of lipid-lowering drugs, we utilized two genetic tools: analysis of polymorphisms affecting the expression levels of drug target genes, and genetic variations linked to low-density lipoprotein cholesterol levels and drug target genes. These variations were sourced from genome-wide association studies (GWAS). We applied Summary-data-based Mendelian Randomization (SMR) and Inverse Variance Weighted Mendelian Randomization (IVW-MR) to gauge the effectiveness of these drugs. RESULTS: Our findings, with SMR results showing an odds ratio (OR) of 1.44 (95% CI: 1.08-1.92; P = 0.011) and IVW-MR results indicating an OR of 1.56 (95% CI: 1.10-2.23; P = 0.013), demonstrate a positive correlation between PCSK9 expression and increased risk of CM. However, no such correlations were observed in other analyses. CONCLUSION: The study concludes that PCSK9 plays a significant role in the development of CM, and its inhibition is linked to a reduced risk of the disease.

Statin administration or blocking PCSK9 alleviates airway hyperresponsiveness and lung fibrosis in high-fat diet-induced obese mice.

BACKGROUND: Obesity is associated with airway hyperresponsiveness and lung fibrosis, which may reduce the effectiveness of standard asthma treatment in individuals suffering from both conditions. Statins and proprotein convertase subtilisin/kexin-9 inhibitors not only reduce serum cholesterol, free fatty acids but also diminish renin-angiotensin system activity and exhibit anti-inflammatory effects. These mechanisms may play a role in mitigating lung pathologies associated with obesity. METHODS: Male C57BL/6 mice were induced to develop obesity through high-fat diet for 16 weeks. Conditional TGF-ß1 transgenic mice were fed a normal diet. These mice were given either atorvastatin or proprotein convertase subtilisin/kexin-9 inhibitor (alirocumab), and the impact on airway hyperresponsiveness and lung pathologies was assessed. RESULTS: High-fat diet-induced obesity enhanced airway hyperresponsiveness, lung fibrosis, macrophages in bronchoalveolar lavage fluid, and pro-inflammatory mediators in the lung. These lipid-lowering agents attenuated airway hyperresponsiveness, macrophages in BALF, lung fibrosis, serum leptin, free fatty acids, TGF-ß1, IL-1ß, IL-6, and IL-17a in the lung. Furthermore, the increased RAS, NLRP3 inflammasome, and cholecystokinin in lung tissue of obese mice were reduced with statin or alirocumab. These agents also suppressed the pro-inflammatory immune responses and lung fibrosis in TGF-ß1 over-expressed transgenic mice with normal diet. CONCLUSIONS: Lipid-lowering treatment has the potential to alleviate obesity-induced airway hyperresponsiveness and lung fibrosis by inhibiting the NLRP3 inflammasome, RAS and cholecystokinin activity.

Association between genetically proxied PCSK9 inhibition and systemic lupus erythematosus risk: A mendelian randomization study.

BACKGROUND: Preclinical and epidemiological studies suggest that proprotein convertase subtilisin/kexin type 9 (PCSK9) had a potential effect on the development of SLE, but it was unclear whether a causal relationship exists. We aimed to investigate the association between genetically proxied inhibition of PCSK9 and the risk of SLE using a two-sample Mendelian randomization (MR) approach. METHODS: Single nucleotide polymorphisms (SNPs) associated with PCSK9 were extracted from pooled data obtained from the Global Lipid Genetics Consortium (GLGC) Genome-wide Association Study (GWAS) related to LDL-c levels, which was used as a proxy for PCSK9 inhibition. Pooled statistics for SLE were obtained from an independent GWAS dataset including 5201 SLE patients and 9066 controls. Inverse variance-weighted random-effects models were used to examine the association between genetically proxied inhibition of PCSK9 and the risk of SLE. MR-Egger, weighted median, weighted mode, Simple mode, and co-location analyses were used as sensitivity analyses to test the robustness of the analyses. RESULTS: Genetically proxied inhibition of PCSK9 was associated with a reduced risk of SLE (OR = 0.51, 95% CI = 0.34 to 0.77, p = .001). This finding was replicated in an earlier GLGC GWAS analysis (OR = 0.59, 95% CI = 0.40 to 0.87, p = .007). Sensitivity analysis ensured that the results were robust. Co-localization analysis did not find evidence of shared causal variation between PCSK9 and SLE. CONCLUSIONS: This Mendelian randomization study showed that PCSK9 was associated with SLE pathogenesis, and its inhibition was associated with a reduced risk of SLE. This study has offered a prospective therapeutic avenue for intervening in the progression of SLE by inhibiting PCSK9 levels.

Optimization of lipid nanoparticles for gene editing of the liver via intraduodenal delivery.

Lipid nanoparticles (LNPs) have recently emerged as successful gene delivery platforms for a diverse array of disease treatments. Efforts to optimize their design for common administration methods such as intravenous injection, intramuscular injection, or inhalation, revolve primarily around the addition of targeting ligands or the choice of ionizable lipid. Here, we employed a multi-step screening method to optimize the type of helper lipid and component ratios in a plasmid DNA (pDNA) LNP library to efficiently deliver pDNA through intraduodenal delivery as an indicative route for oral administration. By addressing different physiological barriers in a stepwise manner, we down-selected effective LNP candidates from a library of over 1000 formulations. Beyond reporter protein expression, we assessed the efficiency in non-viral gene editing in mouse liver mediated by LNPs to knockdown PCSK9 and ANGPTL3 expression, thereby lowering low-density lipoprotein (LDL) cholesterol levels. Utilizing an all-in-one pDNA construct with Strep. pyogenes Cas9 and gRNAs, our results showcased that intraduodenal administration of selected LNPs facilitated targeted gene knockdown in the liver, resulting in a 27% reduction in the serum LDL cholesterol level. This LNP-based all-in-one pDNA-mediated gene editing strategy highlights its potential as an oral therapeutic approach for hypercholesterolemia, opening up new possibilities for DNA-based gene medicine applications.

​Comprehensive mendelian randomization analysis of plasma proteomics to identify new therapeutic targets for the treatment of coronary heart disease and myocardial infarction.

BACKGROUND: Ischemic heart disease is one of the leading causes of mortality worldwide, and thus calls for development of more effective therapeutic strategies. This study aimed to identify potential therapeutic targets for coronary heart disease (CHD) and myocardial infarction (MI) by investigating the causal relationship between plasma proteins and these conditions. METHODS: A two-sample Mendelian randomization (MR) study was performed to evaluate more than 1600 plasma proteins for their causal associations with CHD and MI. The MR findings were further confirmed through Bayesian colocalization, Summary-data-based Mendelian Randomization (SMR), and Transcriptome-Wide Association Studies (TWAS) analyses. Further analyses, including enrichment analysis, single-cell analysis, MR analysis of cardiovascular risk factors, phenome-wide Mendelian Randomization (Phe-MR), and protein-protein interaction (PPI) network construction were conducted to verify the roles of selected causal proteins. RESULTS: Thirteen proteins were causally associated with CHD, seven of which were also causal for MI. Among them, FES and PCSK9 were causal proteins for both diseases as determined by several analytical methods. PCSK9 was a risk factor of CHD (OR = 1.25, 95% CI: 1.13-1.38, P = 7.47E-06) and MI (OR = 1.36, 95% CI: 1.21-1.54, P = 2.30E-07), whereas FES was protective against CHD (OR = 0.68, 95% CI: 0.59-0.79, P = 6.40E-07) and MI (OR = 0.65, 95% CI: 0.54-0.77, P = 5.38E-07). Further validation through enrichment and single-cell analysis confirmed the causal effects of these proteins. Moreover, MR analysis of cardiovascular risk factors, Phe-MR, and PPI network provided insights into the potential drug development based on the proteins. CONCLUSIONS: This study investigated the causal pathways associated with CHD and MI, highlighting the protective and risk roles of FES and PCSK9, respectively. FES. Specifically, the results showed that these proteins are promising therapeutic targets for future drug development.

Exacerbated atherosclerosis in progeria is prevented by progerin elimination in vascular smooth muscle cells but not endothelial cells.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare disease caused by the expression of progerin, a mutant protein that accelerates aging and precipitates death. Given that atherosclerosis complications are the main cause of death in progeria, here, we investigated whether progerin-induced atherosclerosis is prevented in HGPSrev-Cdh5-CreERT2 and HGPSrev-SM22α-Cre mice with progerin suppression in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), respectively. HGPSrev-Cdh5-CreERT2 mice were undistinguishable from HGPSrev mice with ubiquitous progerin expression, in contrast with the ameliorated progeroid phenotype of HGPSrev-SM22α-Cre mice. To study atherosclerosis, we generated atheroprone mouse models by overexpressing a PCSK9 gain-of-function mutant. While HGPSrev-Cdh5-CreERT2 and HGPSrev mice developed a similar level of excessive atherosclerosis, plaque development in HGPSrev-SM22α-Cre mice was reduced to wild-type levels. Our studies demonstrate that progerin suppression in VSMCs, but not in ECs, prevents exacerbated atherosclerosis in progeroid mice.

Dihydrostilbenes and flavonoids from whole plants of Jacobaea vulgaris.

The isolation of previously undescribed 12 compounds from the MeOH extract of Jacobaea vulgaris whole plants is disclosed, comprising 11 dihydrostilbenes (1-11) and one flavanone (12), and eight known compounds (six flavonoids, one dihydrostilbene, and one caffeoylquinic acid). Structural elucidation employed spectroscopic methods, including 1D and 2D NMR spectroscopy, HRESIMS, and ECD calculations. Evaluation of the compounds' effects on PCSK9 and LDLR mRNA expression revealed that compounds 1 and 3 downregulated PCSK9 mRNA while increasing LDLR mRNA expression, suggesting potential cholesterol-lowering properties.

PCSK9 stimulates Syk, PKCδ, and NF-κB, leading to atherosclerosis progression independently of LDL receptor.

Proprotein convertase subtilisin/kexin type-9 (PCSK9) binds to and degrades low-density lipoprotein (LDL) receptor, leading to increase of LDL cholesterol in blood. Its blockers have emerged as promising therapeutics for cardiovascular diseases. Here we show that PCSK9 itself directly induces inflammation and aggravates atherosclerosis independently of the LDL receptor. PCSK9 exacerbates atherosclerosis in LDL receptor knockout mice. Adenylyl cyclase-associated protein 1 (CAP1) is the main binding partner of PCSK9 and indispensable for the inflammatory action of PCSK9, including induction of cytokines, Toll like receptor 4, and scavenger receptors, enhancing the uptake of oxidized LDL. We find spleen tyrosine kinase (Syk) and protein kinase C delta (PKCδ) to be the key mediators of inflammation after PCSK9-CAP1 binding. In human peripheral blood mononuclear cells, serum PCSK9 levels are positively correlated with Syk, PKCδ, and p65 phosphorylation. The CAP1-fragment crystallizable region (CAP1-Fc) mitigates PCSK9-mediated inflammatory signal transduction more than the PCSK9 blocking antibody evolocumab does.

Gene Editing for the Treatment of Hypercholesterolemia.

PURPOSE OF REVIEW: Here, we summarize the key findings from preclinical studies that tested the concept that editing of hepatic genes can lower plasma low-density lipoprotein (LDL)-cholesterol levels to subsequently reduce atherosclerotic cardiovascular disease risk. RECENT FINDINGS: Selective delivery of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing tools targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) to hepatocytes, i.e., through encapsulation into N-acetylgalactosamine-coupled lipid nanoparticles, is able to induce a stable ~ 90% decrease in plasma PCSK9 levels and a concomitant 60% reduction in LDL-cholesterol levels in mice and non-humane primates. Studies in mice have shown that this state-of-the-art technology can be extended to include additional targets related to dyslipidemia such as angiopoietin-like 3 and several apolipoproteins. The use of gene editors holds great promise to lower plasma LDL-cholesterol levels also in the human setting. However, gene editing safety has to be guaranteed before this approach can become a clinical success.

Multifaceted roles of Meg3 in cellular senescence and atherosclerosis.

BACKGROUND AND AIMS: Long noncoding RNAs are involved in the pathogenesis of atherosclerosis. As long noncoding RNAs maternally expressed gene 3 (Meg3) prevents cellular senescence of hepatic vascular endothelium and obesity-induced insulin resistance, we decided to examine its role in cellular senescence and atherosclerosis. METHODS AND RESULTS: By analyzing our data and human and mouse data from the Gene Expression Omnibus database, we found that Meg3 expression was reduced in humans and mice with cardiovascular disease, indicating its potential role in atherosclerosis. In Ldlr-/- mice fed a Western diet for 12 weeks, Meg3 silencing by chemically modified antisense oligonucleotides attenuated the formation of atherosclerotic lesions by 34.9% and 20.1% in male and female mice, respectively, revealed by en-face Oil Red O staining, which did not correlate with changes in plasma lipid profiles. Real-time quantitative PCR analysis of cellular senescence markers p21 and p16 revealed that Meg3 deficiency aggravates hepatic cellular senescence but not cellular senescence at aortic roots. Human Meg3 transgenic mice were generated to examine the role of Meg3 gain-of-function in the development of atherosclerosis induced by PCSK9 overexpression. Meg3 overexpression promotes atherosclerotic lesion formation by 29.2% in Meg3 knock-in mice independent of its effects on lipid profiles. Meg3 overexpression inhibits hepatic cellular senescence, while it promotes aortic cellular senescence likely by impairing mitochondrial function and delaying cell cycle progression. CONCLUSIONS: Our data demonstrate that Meg3 promotes the formation of atherosclerotic lesions independent of its effects on plasma lipid profiles. In addition, Meg3 regulates cellular senescence in a tissue-specific manner during atherosclerosis. Thus, we demonstrated that Meg3 has multifaceted roles in cellular senescence and atherosclerosis.

Evolocumab Treatment in Pediatric Patients With Homozygous Familial Hypercholesterolemia: Pooled Data From Three Open-Label Studies.

BACKGROUND: Pediatric patients with homozygous familial hypercholesterolemia (HoFH) have an increased risk of atherosclerotic cardiovascular disease and difficulty meeting low-density lipoprotein cholesterol (LDL-C) goals. In this post hoc analysis, we evaluated pooled safety and efficacy data from 3 studies in pediatric patients with HoFH treated with the PCSK9 (proprotein convertase subtilisin/kexin type 9) monoclonal antibody inhibitor evolocumab. METHODS: Patients with HoFH aged 10 to 17 years received treatment with open-label evolocumab 420 mg subcutaneously monthly or biweekly in the TAUSSIG, RAMAN, or HAUSER-OLE clinical studies. All patients received background statins with or without ezetimibe. Study duration ranged from 12 to 260 weeks. The primary end point was treatment-emergent adverse events per 100 patient-years. Efficacy end points were changes from baseline to week 12 in lipids and PCSK9. RESULTS: Of the 39 patients in the pooled analysis, 69.2% were males, median age was 13.0 years, and 79.5% (31/39) had genotyped HoFH with LDLR pathogenic variants. Overall, median exposure to evolocumab was 18.2 (Q1, Q3: 3.0, 18.5) months. Treatment-emergent adverse events with an exposure-adjusted patient incidence rate of ≥5% were upper respiratory tract infection (6.6%), influenza (5.2%), and acne (5.0%) per 100 patient-years. Exposure-adjusted patient incidence of serious treatment-emergent adverse events was 13.3% per 100 patient-years. Excluding 4 patients receiving lipoprotein apheresis, week 12 median percentage change from baseline in LDL-C was -2.9% (Q1, Q3: -21.7, 1.5); however, 42.9% (15/35) of patients achieved ≥15% reduction in LDL-C from baseline. Residual LDLR (LDL receptor) activity was not associated with a reduction in LDL-C. CONCLUSIONS: In this pooled data analysis from 3 studies in pediatric patients with HoFH, evolocumab was well tolerated, with no new safety signals reported. These safety findings are consistent with findings from previous studies of evolocumab. Patients showed marked variability in LDL-C reduction. Results from this pooled analysis support guidelines suggesting a trial of PCSK9 inhibitor therapy regardless of estimated residual LDLR function. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT01624142, NCT03403374, and NCT02624869.

Harnessing RNA Interference for Cholesterol Lowering: The Bench-to-Bedside Story of Inclisiran.

Lowering low-density lipoprotein cholesterol (LDL-C) is a cornerstone of reducing risk for atherosclerotic cardiovascular disease. Despite the approval of nonstatin therapies for LDL-C lowering over the past 2 decades, these medications are underused, and most patients are still not at guideline-recommended LDL-C goals. Barriers include poor adherence, clinical inertia, concern for side effects, cost, and complex prior authorization processes. With atherosclerotic cardiovascular disease-related mortality increasing globally, there remains a need for additional therapeutic options for lowering LDL-C as part of an atherosclerotic cardiovascular disease prevention strategy. Following the identification of PCSK9 (proprotein convertase subtilisin/kexin type 9) as a promising therapeutic target, inclisiran was developed using the natural process of RNA interference for robust, sustained prevention of hepatic PCSK9 synthesis. Twice-yearly maintenance subcutaneous inclisiran (following initial loading doses at Day 1 and Day 90) reduces circulating LDL-C levels by ≈50% versus placebo when added to maximally tolerated statins. Long-term safety and tolerability of inclisiran have been assessed, with studies underway to evaluate the effects of inclisiran on cardiovascular outcomes and to provide additional safety and effectiveness data. In 2021, <20 years after the discovery of PCSK9, inclisiran became the first RNA interference therapeutic approved in the United States for LDL-C lowering in patients with established atherosclerotic cardiovascular disease or familial hypercholesterolemia and has since been approved for use in patients with primary hyperlipidemia. This article reviews the journey of inclisiran from bench to bedside, including early development, the clinical trial program, key characteristics of inclisiran, and practical points for its use in the clinic.

CRISPR-Cas9-guided amplification-free genomic diagnosis for familial hypercholesterolemia using nanopore sequencing.

Familial hypercholesterolemia is an inherited disorder that remains underdiagnosed. Conventional genetic testing methods such as next-generation sequencing (NGS) or target PCR are based on the amplification process. Due to the efficiency limits of polymerase and ligase enzymes, these methods usually target short regions and do not detect large mutations straightforwardly. This study combined the long-read nanopore sequencing and CRISPR-Cas9 system to sequence the target DNA molecules without amplification. We originally designed and optimized the CRISPR-RNA panel to target the low-density lipoprotein receptor gene (LDLR) and proprotein convertase subtilisin/kexin type 9 gene (PCSK9) from human genomic DNA followed by nanopore sequencing. The average coverages for LDLR and PCSK9 were 106× and 420×, versus 1.2× for the background genome. Among them, continuous reads were 52x and 307x, respectively, and spanned the entire length of LDLR and PCSK9. We identified pathogenic mutations in both coding and splicing donor regions in LDLR. We also detected an 11,029 bp large deletion in another case. Furthermore, using continuous long reads generated from the benchmark experiment, we demonstrated how a false-positive 670 bp deletion caused by PCR amplification errors was easily eliminated.