LDLR Variants Research Articles

Novel Finnish-enriched variants causing severe hypercholesterolemia and their clinical impact on coronary artery disease

Background and aimsSevere hypercholesterolemia (LDL-cholesterol ≥ 5 mmol/l) is a major risk factor for coronary artery disease (CAD). The etiology incudes both genetic and nongenetic factors, but persons carrying mutations in known hypercholesterolemia-associated genes are at significantly higher CAD risk than non-carriers. Yet, a significant proportion of mutation carriers remains undetected while the assessment of genetic candidate variants in clinical practice is challenging. MethodsTo address these challenges, we set out to test the utility of a practical approach to leverage data from a large reference cohort, the FinnGen Study encompassing 356,082 persons with extensive longitudinal health record information, to aid the clinical evaluation of single genetic candidate genes variants detected by exome sequence analysis in a target population of 351 persons with severe hypercholesterolemia. ResultsWe identified 23 rare missense mutations in known hypercholesterolemia genes, 3 of which were previously described mutations (LDLR Pro309Lysfs, LDLR Arg595Gln and APOB Arg3527Gln). Subsequent in silico and clinical assessment of the remaining 20 variants pinpointed two likely hypercholesterolemia-associated variants in LDLR (Arg574Leu and Glu626Lys) and one in LDLRAP1 (Arg151Trp). Heterozygous carriers of the novel LDLR and LDLRAP1 variants received statin treatment more often than non-carriers (OR 2.1, p = 1.8e-6 and OR 1.4, p = 0.001) and untreated carriers had higher risk for ischemic heart disease (OR 2.0, p = 0.03 and OR 1.8, p = 0.008). ConclusionsOur data elucidate the wide spectrum of genetic variants impacting hypercholesterolemia and demonstrate the utility of a large reference population to assess the heterogeneous impact of candidate gene variants on cardiovascular disease risk.

B-252 In Vitro Expression Analysis of Variants in the Upstream Region of Genes Related to Familial Hypercholesterolemia

Abstract Background Familial hypercholesterolemia (FH) is commonly described as an autosomal dominant disorder caused mainly by variants in genes LDLR, APOB, and PCSK9, resulting in severe hypercholesterolemia. However, the substantial number of individuals who are clinically diagnosed but negative for known FH-causing variants indicates that relevant variants outside of frequently analyzed regions might exist. Considering the increasing number of promoter variants reported as likely to cause FH and the relevance of in vitro expression assays in the pathogenicity evaluation of these variants, this work aimed to examine the functionality of variants LDLR rs36218923-T and APOB rs934197-T, identified in a Brazilian FH cohort. Methods Upstream fragments of LDLR (-861 bp to +85 bp) and APOB (-1460 bp to +127 bp) were PCR amplified from genomic DNA of two FH patients carrying LDLR rs36218923-T and APOB rs934197-T in heterozygosis and homozygosis, respectively. After linearization and PGK promoter removal of the pGL4.53 vector using Kpn I and Hind III restriction enzymes, fragments were cloned upstream of the Firefly Luciferase (luc2) coding region using the NEBuilder HiFi DNA Assembly Cloning Kit. Site-directed mutagenesis was also performed using partially overlapping primers with 3´-overhangs and Platinum SuperFi II DNA Polymerase to produce different constructs, including one containing LDLR rs879254375-G, which is a known FH-causing variant. All constructs were confirmed by Sanger sequencing. pGL4.53 constructs containing either wild type or variant alleles and pNL1.1.TK (control of transfection variation, expressing NanoLuc Luciferase) were co-transfected into HepG2 cells. Wild type constructs were considered positive controls, while promoterless pGL4.53 was considered negative control. Luciferase assays were carried out 48 h after transfection using the Nano-Glo Dual-Luciferase Reporter Assay System. After normalization of luminescence units of each assay (Firefly/Nanoluc), luciferase activity was determined as the mean of four transfections with the assay performed in triplicate. Results The construct carrying LDLR rs36218923-T variant caused a significant mean reduction of luciferase activity (75,4% ± 2,9% SEM) over the promoterless construct relative to LDLR wild type construct. However, the construct harboring the LDLR rs879254375-G variant caused a more substantial reduction of luciferase activity relative to the wild type construct (91,2% ± 0,9% SEM). This reduction was consistent with shown by the previous study that reported LDLR rs879254375-G as a functional variant, which corroborates the methods adopted in this study. APOB rs934197-T was previously reported as functional based on a CAT assay using constructs of two tandemly arranged 30 bp fragments of APOB promoter, but in our study, the construct carrying the entire APOB promoter sequence and harboring rs934197-T variant did not cause a significant difference in mean luciferase activity compared to APOB wild type construct (P = 0.564). It is important to mention that our APOB upstream constructs had the additional variants rs617314-G, rs1560357-T, rs1625764-A, and rs1800481-G, which are considered benign because they occur at high frequency in the general population (0.82-1). Conclusion Our results suggest that LDLR rs36218923-T is likely to contribute to FH phenotype and emphasize the importance of performing in vitro expression assays in determining relevant upstream variants for FH molecular characterization.

Effects of LDLR variants rs5928, rs750518671 and rs879254797 on protein structure and functional activity in HepG2 cells transfected with CRISPR/Cas9 constructs

Familial Hypercholesterolemia (FH) is a genetic disorder associated with premature atherosclerosis and increased risk of cardiovascular diseases. LDLR deleterious mutations are associated with FH, however the role of some missense variants in FH pathogenicity remains to be elucidated. This study explored the predictive impact of LDLR missense variants on protein structure and investigated their functional effects on LDLR expression in HepG2 cells transfected with CRISPR/Cas9 constructs. FH (n = 287) and non-FH patients (n = 45) were selected, and lipid profile was obtained from medical records. LDLR variants were identified using an exon-targeted gene sequencing strategy, considering its cost-effective to increase accuracy in the identification step of the most likely FH-related variants in a less laborious process. LDLR variants were selected based on conflicting pathogenicity results found in Clinvar, in silico prediction tools, affected LDLR domains, and less common variants considering minor allele frequency < 0.05. Molecular modeling studies were used to predict the effects of LDLR missense variants on protein structure. Recombinant LDLR variants were constructed using CRISPR/Cas9 system and were used to transfect HepG2 cells. Functional assays in transfected cells were performed to assess LDLR expression using flow cytometry and western blotting, and LDLR activity using flow cytometry and confocal microscopy. The variants rs121908039 (c.551G>A, p.C184Y), rs879254797 (c.1118G>A, p.G373D), rs28941776 (c.1646G>A, p.G549D), rs750518671 (c.2389G>C, p.V797L), rs5928 (c.2441G>A, p.R814Q) and rs137853964 (c.2479G>A, p.V827I) were selected for molecular docking analysis. The p.C184Y exhibited a favorable energy change for protein stability due to its interaction with EGF-A/EGF-B regions; p.G373D and p.G549D displayed intermediate energy changes; and p.R814Q and p.V827I showed smaller energy changes. The results of functional assays showed that p.G373D, p.V797L and p.R814Q reduced LDLR expression and activity (p < 0.05). Microscopic analysis of the p.V797L and p.G373D variants revealed altered lipid localization and accumulation in transfected HepG2 cells. Carriers of p.G549D, p.V797L and p.R814Q had higher LDL cholesterol levels than non-FH group, and (p < 0.05). p.G373D and p.G549D were associated with clinical manifestations of FH. In conclusion, the p.C184Y, p.G373D, p.G549D and p.R814Q variants alter protein stability and intramolecular interactions, while p.V797L has a minimal impact on protein stability, and p.V827I has no significant intramolecular interactions. p.G373D, p.V767L and p.R814Q are associated with impaired LDLR expression and activity.

Low-Cost High-Throughput Genotyping for Diagnosing Familial Hypercholesterolemia.

Familial hypercholesterolemia (FH) is a common but underdiagnosed genetic disorder characterized by high low-density lipoprotein cholesterol levels and premature cardiovascular disease. Current sequencing methods to diagnose FH are expensive and time-consuming. In this study, we evaluated the accuracy of a low-cost, high-throughput genotyping array for diagnosing FH. An Illumina Global Screening Array was customized to include probes for 636 variants, previously classified as FH-causing variants. First, its theoretical coverage was assessed in all FH variant carriers diagnosed through next-generation sequencing between 2016 and 2022 in the Netherlands (n=1772). Next, the performance of the array was validated in another sample of FH variant carriers previously identified in the Dutch FH cascade screening program (n=1268). The theoretical coverage of the array for FH-causing variants was 91.3%. Validation of the array was assessed in a sample of 1268 carriers of whom 1015 carried a variant in LDLR, 250 in APOB, and 3 in PCSK9. The overall sensitivity was 94.7% and increased to 98.2% after excluding participants with variants not included in the array design. Copy number variation analysis yielded a 89.4% sensitivity. In 18 carriers, the array identified a total of 19 additional FH-causing variants. Subsequent DNA analysis confirmed 5 of the additionally identified variants, yielding a false-positive result in 16 subjects (1.3%). The FH genotyping array is a promising tool for genetically diagnosing FH at low costs and has the potential to greatly increase accessibility to genetic testing for FH. Continuous customization of the array will further improve its performance.

Polygenic Risk, Rare Variants, and Family History: Independent and Additive Effects on Coronary Heart Disease

BackgroundGenetic factors are not included in prediction models for coronary heart disease (CHD). ObjectivesThe authors assessed the predictive utility of a polygenic risk score (PRS) for CHD (defined as myocardial infarction, coronary revascularization, or cardiovascular death) and whether the risks due to monogenic familial hypercholesterolemia (FH) and family history (FamHx) are independent of and additive to the PRS. MethodsIn UK-biobank participants, PRSCHD was calculated using metaGRS, and 10-year risk for incident CHD was estimated using the pooled cohort equations (PCE). The area under the curve (AUC) of the receiver operator curve and net reclassification improvement (NRI) were assessed. FH was defined as the presence of a pathogenic or likely pathogenic variant in LDLR, APOB, or PCSK9. FamHx was defined as a diagnosis of CHD in first-degree relatives. Independent and additive effects of PRSCHD, FH, and FamHx were evaluated in stratified analyses. ResultsIn 323,373 participants with genotype data, the addition of PRSCHD to PCE increased the AUC from 0.759 (95% CI: 0.755-0.763) to 0.773 (95% CI: 0.769-0.777). The AUC and NRIEvent for PRSCHD were higher before the age of 55 years. Of 199,997 participants with exome sequence data, 10,000 had a PRSCHD ≥95th percentile (PRSP95), 673 had FH, and 46,163 had FamHx. The CHD risk associated with PRSP95 was independent of FH and FamHx. The risks associated with combinations of PRSCHD, FH, and FamHx were additive and comprehensive estimates could be obtained by multiplying the risk from each genetic factor. ConclusionsIncorporating PRSCHD into the PCE improves risk prediction for CHD, especially at younger ages. The associations of PRSCHD, FH, and FamHx with CHD were independent and additive.

Resolving conflicting LDLR variants in ClinVar - Progress of the ClinGen familial hypercholesterolemia variant curation expert panel

Resolving conflicting LDLR variants in ClinVar - Progress of the ClinGen familial hypercholesterolemia variant curation expert panel

Filling the gap: Genetic risk assessment in hypercholesterolemia using LDL-C and LPA genetic scores.

Routine genetic testing in hypercholesterolemia patients reveals a causative monogenic variant in less than 50% of affected individuals. Incomplete genetic characterization is partly due to polygenic factors influencing low-density-lipoprotein-cholesterol (LDL-C). Additionally, functional variants in the LPA gene affect lipoprotein(a)-associated cholesterol concentrations but are difficult to determine due to the complex structure of the LPA gene. In this study we examined whether complementing standard sequencing with the analysis of genetic scores associated with LDL-C and Lp(a) concentrations improves the diagnostic output in hypercholesterolemia patients. 1.020 individuals including 252 clinically diagnosed hypercholesterolemia patients from the FH Register Austria were analyzed by massive-parallel-sequencing of candidate genes combined with array genotyping, identifying nine novel variants in LDLR. For each individual, validated genetic scores associated with elevated LDL-C and Lp(a) were calculated based on imputed genotypes. Integrating these scores especially the score for Lp(a) increased the proportion of individuals with a clearly defined disease etiology to 68.8% compared to 46.6% in standard genetic testing. The study highlights the major role of Lp(a) in disease etiology in clinically diagnosed hypercholesterolemia patients, of which parts are misclassified. Screening for monogenic causes of hypercholesterolemia and genetic scores for LDL-C and Lp(a) permits more precise diagnosis, allowing individualized treatment.

High-Throughput Microscopy Characterization of Rare LDLR Variants

Familial hypercholesterolemia (FH) is the most common inherited life-threatening disorder of lipid metabolism. Early diagnosis and treatment are the key to reduce the cumulative life-long cardiovascular burden of patients with FH. The high number of LDLR variants described as variants of unknown significance is the largest obstacle to achieve a definitive FH diagnosis. This study established a time- and cost-effective high-throughput cell-based assay to functionally profile LDLR variants, which allowed us to discriminate disruptive rare variants from silent ones. This work generated a valuable resource for systematic functional characterization of LDLR variants solving 1 of the major issues to achieve a definitive FH diagnosis.

Cholesterol Contributes to Risk, Severity, and Machine Learning-Driven Diagnosis of Lyme Disease.

Lyme disease is the most prevalent vector-borne disease in the US, yet its host factors are poorly understood and diagnostic tests are limited. We evaluated patients in a large health system to uncover cholesterol's role in the susceptibility, severity, and machine learning-based diagnosis of Lyme disease. A longitudinal health system cohort comprised 1 019 175 individuals with electronic health record data and 50 329 with linked genetic data. Associations of blood cholesterol level, cholesterol genetic scores comprising common genetic variants, and burden of rare loss-of-function (LoF) variants in cholesterol metabolism genes with Lyme disease were investigated. A portable machine learning model was constructed and tested to predict Lyme disease using routine lipid and clinical measurements. There were 3832 cases of Lyme disease. Increasing cholesterol was associated with greater risk of Lyme disease and hypercholesterolemia was more prevalent in Lyme disease cases than in controls. Cholesterol genetic scores and rare LoF variants in CD36 and LDLR were associated with Lyme disease risk. Serological profiling of cases revealed parallel trajectories of rising cholesterol and immunoglobulin levels over the disease course, including marked increases in individuals with LoF variants and high cholesterol genetic scores. The machine learning model predicted Lyme disease solely using routine lipid panel, blood count, and metabolic measurements. These results demonstrate the value of large-scale genetic and clinical data to reveal host factors underlying infectious disease biology, risk, and prognosis and the potential for their clinical translation to machine learning diagnostics that do not need specialized assays.

Identification of pathogenic variants in the Brazilian cohort with Familial hypercholesterolemia using exon-targeted gene sequencing

Familial hypercholesterolemia (FH) is a monogenic disease characterized by high plasma low-density lipoprotein cholesterol (LDL-c) levels and increased risk of premature atherosclerotic cardiovascular disease. Mutations in FH‐related genes account for 40% of FH cases worldwide. In this study, we aimed to assess the pathogenic variants in FH-related genes in the Brazilian FH cohort FHBGEP using exon-targeted gene sequencing (ETGS) strategy. FH patients (n = 210) were enrolled at five clinical sites and peripheral blood samples were obtained for laboratory testing and genomic DNA extraction. ETGS was performed using MiSeq platform (Illumina). To identify deleterious variants in LDLR, APOB, PCSK9, and LDLRAP1, the long-reads were subjected to Burrows-Wheeler Aligner (BWA) for alignment and mapping, followed by variant calling using Genome Analysis Toolkit (GATK) and ANNOVAR for variant annotation. The variants were further filtered using in-house custom scripts and classified according to the American College Medical Genetics and Genomics (ACMG) guidelines. A total of 174 variants were identified including 85 missense, 3 stop-gain, 9 splice-site, 6 InDel, and 71 in regulatory regions (3′UTR and 5′UTR). Fifty-two patients (24.7%) had 30 known pathogenic or likely pathogenic variants in FH-related genes according to the American College Medical and Genetics and Genomics guidelines. Fifty-three known variants were classified as benign, or likely benign and 87 known variants have shown uncertain significance. Four novel variants were discovered and classified as such due to their absence in existing databases. In conclusion, ETGS and in silico prediction studies are useful tools for screening deleterious variants and identification of novel variants in FH-related genes, they also contribute to the molecular diagnosis in the FHBGEP cohort.

Identification of an LDLR variant in a Chinese familial hypercholesterolemia and its relation to ROS/NLRP3-Mediated pyroptosis in hepatic cells.

Familial hypercholesterolemia (FH) is a common autosomal dominant hereditary disease. Its early diagnosis and intervention significantly improve the patient's quality of life. However, there are few types of research on the FH pathogenic genes in China. In this study, we recruited a family diagnosed with FH and used whole exome sequencing (WES) to analyze the proband variants. Intracellular cholesterol level, reactive oxygen species (ROS) level, and the expression of pyroptosis-related genes were detected after overexpression of wild-type or variant LDLR in L02 cells. A heterozygous missense variant predicted to be deleterious to LDLR (c.1879G > A, p.Ala627Thr) was identified in the proband. Mechanistically, intracellular cholesterol level, ROS level, and the expression of pyroptosis-related genes, nucleotide-binding oligomerization domain-like receptor family protein 3 (NLRP3) inflammasome and components (caspase 1, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and NLRP3), gasdermin D (GSDMD), interleukin (IL) -18, IL-1β was elevated in the variant LDLR group, which was attenuated by inhibition of ROS. FH is associated with a variant (c.1879G>A, p.Ala627Thr) in the LDLR gene. Regarding the mechanism, the ROS/NLRP3-mediated pyroptosis in hepatic cells may contribute to the pathogenesis of the LDLR variant.

Genetic Testing Enables the Diagnosis of Familial Hypercholesterolemia Underdiagnosed by Clinical Criteria: Analysis of Japanese Early-Onset Coronary Artery Disease Patients.

Definitive diagnosis of familial hypercholesterolemia (FH) is paramount for the risk management of patients and their relatives. The present study aimed to investigate the frequency of gene variants contributing to low-density lipoprotein cholesterol (LDL-C) metabolism and their clinical relevance in patients with early-onset coronary artery disease (EOCAD). Among 63 consecutive patients with EOCAD (men <55 years or women <65 years) who underwent percutaneous coronary intervention (PCI) from 2013 to 2019 at Keio University Hospital, 52 consented to participate in this retrospective study. Targeted sequencing of LDLR, PCSK9, APOB, and LDLRAP1 was performed. Of the 52 patients enrolled (42 men; mean age: 50 ± 6 years), one (LDLR, c.1221_1222delCGinsT) harbored a pathogenic mutation, and one (APOB, c.10591A>G) harbored variants of uncertain significance. Both the patients harboring the variants were male, showing no history of diabetes mellitus or chronic kidney disease, no family history of EOCAD, and no physical findings of FH (i.e., tendon xanthomas or Achilles tendon thickening). Patients harboring the LDLR variant had three-vessel disease, were on a statin prescription at baseline, and had stable LDL-C levels; however, the case showed a poor response to the intensification of medication after PCI. Approximately 3.8% of patients with EOCAD harbored variants of gene related to LDL-C metabolism; there were no notable indicators in the patients' background or clinical course to diagnose FH. Given the difficulty in diagnosing FH based on clinical manifestations and family history, genetic testing could enable the identification of hidden risk factors and provide early warnings to their relatives.

Additive Effect of APOE Rare Variants on the Phenotype of Familial Hypercholesterolemia.

Autosomal dominant hypercholesterolemia (ADH) is due to deleterious variants in LDLR, APOB, or PCSK9 genes. Double heterozygote for these genes induces a more severe phenotype. More recently, a new causative variant of heterozygous ADH was identified in APOE. Here we study the phenotype of 21 adult patients, double heterozygotes for rare LDLR and rare APOE variants (LDLR+APOE) in a national wide French cohort. LDLR, APOB, PCSK9, and APOE genes were sequenced in 5743 probands addressed for ADH genotyping. The lipid profile and occurrence of premature atherosclerotic cardiovascular diseases were compared between the LDLR+APOE carriers (n=21) and the carriers of the same LDLR causative variants alone (n=22). The prevalence of LDLR+APOE carriers in this French ADH cohort is 0.4%. Overall, LDL (low-density lipoprotein)-cholesterol concentrations were 23% higher in LDLR+APOE patients than in LDLR patients (9.14±2.51 versus 7.43±1.59 mmol/L, P=0.0221). When only deleterious or probably deleterious variants were considered, the LDL-cholesterol concentrations were 46% higher in LDLR+APOE carriers than in LDLR carriers (10.83±3.45 versus 7.43±1.59 mmol/L, P=0.0270). Two patients exhibited a homozygous familial hypercholesterolemia phenotype (LDL-cholesterol >13 mmol/L). Premature atherosclerotic cardiovascular disease was more common in LDLR+APOE patients than in LDLR carriers (70% versus 30%, P=0.026). Although an incomplete penetrance should be taken into account for APOE variant classification, these results suggest an additive effect of deleterious APOE variants on ADH phenotype highlighting the relevance of APOE sequencing.

Identification and Functional Analysis of APOB Variants in a Cohort of Hypercholesterolemic Patients.

Mutations in APOB are the second most frequent cause of familial hypercholesterolemia (FH). APOB is highly polymorphic, and many variants are benign or of uncertain significance, so functional analysis is necessary to ascertain their pathogenicity. Our aim was to identify and characterize APOB variants in patients with hypercholesterolemia. Index patients (n = 825) with clinically suspected FH were analyzed using next-generation sequencing. In total, 40% of the patients presented a variant in LDLR, APOB, PCSK9 or LDLRAP1, with 12% of the variants in APOB. These variants showed frequencies in the general population lower than 0.5% and were classified as damaging and/or probably damaging by 3 or more predictors of pathogenicity. The variants c.10030A>G;p.(Lys3344Glu) and c.11401T>A;p.(Ser3801Thr) were characterized. The p.(Lys3344Glu) variant co-segregated with high low-density lipoprotein (LDL)-cholesterol in 2 families studied. LDL isolated from apoB p.(Lys3344Glu) heterozygous patients showed reduced ability to compete with fluorescently-labelled LDL for cellular binding and uptake compared with control LDL and was markedly deficient in supporting U937 cell proliferation. LDL that was carrying apoB p.(Ser3801Thr) was not defective in competing with control LDL for cellular binding and uptake. We conclude that the apoB p.(Lys3344Glu) variant is defective in the interaction with the LDL receptor and is causative of FH, whereas the apoB p.(Ser3801Thr) variant is benign.

Genotypic and phenotypic features of dyslipidemia in a sample of pediatric patients in China

BackgroundDyslipidemia, especially hypercholesterolemia is of significant clinical interest. Precise diagnosis is not paid enough attention to about the management of pediatric patients with hypercholesterolemia, which is especially apparent in China. Given this, we designed this study to confirm the specific molecular defects associated with hypercholesterolemia using whole-exome sequencing (WES) to be helpful for precise diagnosis and treatment.MethodsPediatric patients were enrolled using specific criteria and their clinical information were recorded for later evaluation in conjunction with the WES completed for each of these patients.ResultsOur criteria allowed for the initial enrollment of 35 patients, 30 of whom (aged 1.02–12.99 years) underwent successful genetic sequencing and clinical investment. Positive results were obtained in 63.33% (19/30) of these patients. We identified 25 variants in 30 pediatric patients with persistent hypercholesterolemia, seven of them were novel and variants in LDLR and ABCG5/ABCG8 ranks first and second, respectively. Further analysis revealed that the levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), apolipoprotein B (ApoB) and lipoprotein (a) were higher in patients with positive genetic results.ConclusionOur study enriched the genetic and phenotypic spectra for hypercholesterolemia in young patients. Genetic testing is important for the prognostics and treatment of pediatric patients. Heterozygous ABCG5/8 variants may be underestimated in pediatric patients with hypercholesterolemia.

Genetic Identification of Homozygous Familial Hypercholesterolemia by Long-Read Sequencing Among Patients With Clinically Diagnosed Heterozygous Familial Hypercholesterolemia.

Homozygous familial hypercholesterolemia (HoFH) is a rare genetic disorder characterized by extremely elevated plasma LDL-C (low-density lipoprotein cholesterol) and accelerated atherosclerosis. Accurate identification of patients with HoFH is essential as they may be eligible for specialized treatments. We hypothesized that a subset of patients with clinically diagnosed heterozygous FH (HeFH) may in fact have HoFH, and this could be identified by genetic diagnosis. We recruited patients with a clinical diagnosis of HeFH based on a Dutch Lipid Clinic Network score ≥6 and no secondary cause of hypercholesterolemia. We performed targeted next-generation sequencing of the LDLR, APOB, PCSK9, and LDLRAP1 genes, followed by long-read sequencing of the LDLR gene in patients with >1 pathogenic LDLR variant. We examined lipid levels and cardiovascular events. Among 705 patients with clinically diagnosed HeFH, we identified a single pathogenic variant in 300 (42.6%) and >1 pathogenic variant in the LDLR gene in 11 patients (1.6%). We established a genetic diagnosis of HoFH in 6 (0.9%) patients (3 true homozygotes and 3 compound heterozygotes). The mean baseline LDL-C and prevalence of premature cardiovascular disease of patients with genetically identified HoFH was significantly higher than patients with HeFH. In a cohort of patients with clinically diagnosed HeFH, genetic testing including long-read sequencing revealed that 0.9% had HoFH. These patients tended to have a more severe clinical phenotype. Genetic testing of patients with clinical FH may identify patients with HoFH that had eluded clinical diagnosis.

Opportunistic Genetic Screening for Familial Hypercholesterolemia in Heart Transplant Patients.

Heart transplantation remains the gold standard for the treatment of advanced heart failure (HF). Identification of the etiology of HF is mandatory, as the specific pathology can determine subsequent treatment. Early identification of familial hypercholesterolemia (FH), the most common genetic disorder associated with premature cardiovascular disease, has a potential important impact on clinical management and public health. We evaluated the genetic information in the genes associated with FH in a cohort of 140 heart-transplanted patients. All patients underwent NGS genetic testing including LDLR, APOB, and PCSK9. We identified four carriers of rare pathogenic variants in LDLR and APOB. Although all four identified carriers had dyslipidemia, only the one carrying the pathogenic variant LDLR c.676T>C was transplanted due to CAD. Another patient with heart valvular disease was carrier of the controversial LDLR c.2096C>T. Two additional patients with non-ischemic dilated cardiomyopathy were carriers of variants in APOB (c.4672A>G and c.5600G>A). In our cohort, we identified the genetic cause of FH in patients that otherwise would not have been diagnosed. Opportunistic genetic testing for FH provides important information to perform personalized medicine and risk stratification not only for patients but also for relatives at concealed high cardiovascular risk. Including the LDLR gene in standard NGS cardiovascular diagnostics panels should be considered.

Large-Scale CRISPR Screen of LDLR Pathogenic Variants.

Familial hypercholesterolemia (FH) is a frequently occurring genetic disorder that is linked to early-onset cardiovascular disease. If left untreated, patients with this condition can develop severe cardiovascular complications. Unfortunately, many patients remain undiagnosed, and even when diagnosed, the treatment is often not optimal. Although mutations in the LDLR gene are the primary cause of FH, predicting whether novel variants are pathogenic is not a straightforward task. Understanding the functionality of LDLR variants is crucial in uncovering the genetic basis of FH. Our study utilized CRISPR/Cas9 cytosine base editors in pooled screens to establish a novel approach for functionally assessing tens of thousands of LDLR variants on a large scale. A total of more than 100 single guide RNAs (sgRNAs) targeting LDLR pathogenic mutations were successfully screened with relatively high accuracy. Out of these, 5 sgRNAs were further subjected to functional verification studies, including 1 in the promoter, 1 in the antisense RNA, 1 in the exon, and 2 in the intron. Except for the variant caused by the sgRNA located at intron 16, the functionalities of the other LDLR variants were all downregulated. The high similarity of LDLR intron sequences may lead to some false positives. Overall, these results confirm the reliability of the large-scale screening strategy for functional analysis of LDLR variants, and the screened candidate pathogenic mutations could be used as an auxiliary means of clinical gene detection to prevent FH-induced heart disease.

LDLR missense variants disturb structural conformation and LDLR activity in T-lymphocytes of Familial hypercholesterolemia patients

Familial hypercholesterolemia (FH) is caused by deleterious mutations in the LDLR that increase markedly low-density lipoprotein (LDL) cholesterol and cause premature atherosclerotic cardiovascular disease. Functional effects of pathogenic LDLR variants identified in Brazilian FH patients were assessed using in vitro and in silico studies. Variants in LDLR and other FH-related genes were detected by exon-target gene sequencing. T-lymphocytes were isolated from 26 FH patients, and 3 healthy controls and LDLR expression and activity were assessed by flow cytometry and confocal microscopy. The impact of LDLR missense variants on protein structure was assessed by molecular modeling analysis. Ten pathogenic or likely pathogenic LDLR variants (six missense, two stop-gain, one frameshift, and one in splicing region) and six non-pathogenic variants were identified. Carriers of pathogenic and non-pathogenic variants had lower LDL binding and uptake in activated T-lymphocytes compared to controls (p < 0.05), but these variants did not influence LDLR expression on cell surface. Reduced LDL binding and uptake was also observed in carriers of LDLR null and defective variants. Modeling analysis showed that p.(Ala431Thr), p.(Gly549Asp) and p.(Gly592Glu) disturb intramolecular interactions of LDLR, and p.(Gly373Asp) and p.(Ile488Thr) reduce the stability of the LDLR protein. Docking and molecular interactions analyses showed that p.(Cys184Tyr) and p.(Gly373Asp) alter interaction of LDLR with Apolipoprotein B (ApoB). In conclusion, LDLR null and defective variants reduce LDL binding capacity and uptake in activated T-lymphocytes of FH patients and LDLR missense variants affect LDLR conformational stability and dissociation of the LDLR-ApoB complex, having a potential role in FH pathogenesis.

Abstract 10360: Polygenic Risk Score, Rare Monogenic Variants, and Family History Have Independent and Additive Effects on the Risk of Coronary Heart Disease

Introduction. We assessed the effects of a polygenic risk score (PRS), monogenic variants (Mono + ), and family history (FamHx) on the risk of coronary heart disease (CHD) and whether incorporating a PRS into the Pooled Cohort Equations (PCE) improves risk prediction. Method. In 199,997 UK Biobank participants, we calculated a PRS CHD (metaGRS, based on ~1.6 million single nucleotide variants), identified individuals with monogenic etiology (presence of a pathogenic variant in LDLR , APOB , or PCSK9 ), and ascertained family history of CHD (CHD in a first-degree relative). Odds of CHD (myocardial infarction/ coronary revascularization/ cardiovascular death) associated with PRS CHD , Mono + , and FamHx at age 55 were computed using logistic regression analyses adjusted for age, sex, and first 4 principal components of ancestry. We combined PRS CHD and PCE into an integrated score (IS) and assessed net reclassification using an actionable risk threshold of 7.5%. Results. The odds ratio (OR) for CHD in the top 5 th percentile of PRS CHD (PRS P95 ), Mono + , and FamHx were 3.37 (2.98 - 3.81), 2.68 (1.67 - 4.07), and 1.61 (1.47 - 1.76), respectively (all p&lt;0.001). These effects were independent and similar in different subgroups. ( Figure 1-A ) Compared to PRS Q2-4 Mono - (3 middle quintiles of PRS CHD and no monogenic variants), the OR for CHD ranged from 1.73 (0.28 - 5.48, p = 0.446) in PRS Q1 Mono + to 5.10 (2.14 - 10.24, p &lt;0.001) in PRS Q5 Mono + . A similar pattern was seen for other combinations, indicating additive effects ( Figure 1-B ). Incorporating PRS CHD into PCE resulted in a net reclassification improvement of 4.56%. Compared to those with high PCE and low IS, the hazard ratio for CHD in those with low PCE and high IS was 1.48 (1.34 - 1.64, p &lt;0.001). Conclusion. The CHD risk associated with PRS CHD , monogenic variants, and family history is independent and additive. Combining PRS CHD and PCE improved risk prediction, that could improve further by adding monogenic variants and family history.