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.

Lipidomic analysis identified potential predictive biomarkers of statin response in subjects with Familial hypercholesterolemia

Familial hypercholesterolemia (FH) is a disorder of lipid metabolism that causes elevated low-density lipoprotein cholesterol (LDL-c) and increased premature atherosclerosis risk. Statins inhibit endogenous cholesterol biosynthesis, which reduces LDL-c plasma levels and prevent from cardiovascular events. This study aimed to explore the effects of statin treatment on serum lipidomic profile and to identify biomarkers of response in subjects with FH. Seventeen adult FH patients underwent a 6-week washout followed by 4-week treatment with atorvastatin (80 mg/day) or rosuvastatin (40 mg/day). LDL-c response was considered good (40­70 % reduction, n = 9) or poor (3­33 % reduction, n = 8). Serum lipidomic profile was analyzed by ultra-high-performance liquid chromatography combined with electrospray ionization tandem time-of-flight mass spectrometry, and data were analyzed using MetaboAnalyst v5.0. Lipidomic analysis identified 353 lipids grouped into 16 classes. Statin treatment reduced drastically 8 of 13 lipid classes, generating a characteristic lipidomic profile with a significant contribution of phosphatidylinositols (PI) 16:0/18:2, 18:0/18:1 and 18:0/18:2; and triacylglycerols (TAG) 18:2x2/18:3, 18:1/18:2/18:3, 16:1/18:2x2, 16:1/18:2/18:3 and 16:1/18:2/Arachidonic acid (p-adjusted <0.05). Biomarker analysis implemented in MetaboAnalyst subsequently identified PI 16:1/18:0, 16:0/18:2 and 18:0/18:2 as predictors of statin response with and receiver operating characteristic (ROC) areas under the curve of 0.98, 0.94 and 0.91, respectively. In conclusion, statins extensively modulate the overall serum lipid composition of FH individuals and these findings suggest that phosphatidyl-inositol molecules are potential predictive biomarkers of statin response.

Influence of polymorphisms in IRS1, IRS2, MC3R, and MC4R on metabolic and inflammatory status and food intake in Brazilian adults: An exploratory pilot study

Polymorphisms in genes of leptin-melanocortin and insulin pathways have been associated with obesity and type 2 diabetes. We hypothesized that polymorphisms in IRS1, IRS2, MC3R, and MC4R influence metabolic and inflammatory markers and food intake composition in Brazilian subjects. This exploratory pilot study included 358 adult subjects. Clinical, anthropometric, and laboratory data were obtained through interview and access to medical records. The variants IRS1 rs2943634 A˃C, IRS2 rs1865434 C>T, MC3R rs3746619 C>A, and MC4R rs17782313 T>C were analyzed by real-time polymerase chain reaction. Food intake composition was assessed in a group of subjects with obesity (n = 84) before and after a short-term nutritional counseling program (9 weeks). MC4R rs17782313 was associated with increased risk of obesity (P = .034). Multivariate linear regression analysis adjusted by covariates indicated associations of IRS2 rs1865434 with reduced low-density lipoprotein cholesterol and resistin, MC3R rs3746619 with high glycated hemoglobin, and IRS1 rs2943634 and MC4R rs17782313 with increased high-sensitivity C-reactive protein (P < .05). Energy intake and carbohydrate and total fat intakes were reduced after the diet-oriented program (P < .05). Multivariate linear regression analysis showed associations of IRS2 rs1865434 with high basal fiber intake, IRS1 rs2943634 with low postprogram carbohydrate intake, and MC4R rs17782313 with low postprogram total fat and saturated fatty acid intakes (P < .05). Although significant associations did not survive correction for multiple comparisons using the Benjamini-Hochberg method in this exploratory study, polymorphisms in IRS1, IRS2, MC3R, and MC4R influence metabolic and inflammatory status in Brazilian adults. IRS1 and MC4R variants may influence carbohydrate, total fat, and saturated fatty acid intakes in response to a diet-oriented program in subjects with obesity.

Lipidomic analysis identified potential predictive biomarkers of statin response in subjects with Familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is a disorder of lipid metabolism that causes elevated low-density lipoprotein cholesterol (LDL-c) and increased premature atherosclerosis risk. Statins inhibit endogenous cholesterol biosynthesis, which reduces LDL-c plasma levels and prevent from cardiovascular events. This study aimed to explore the effects of statin treatment on serum lipidomic profile and to identify biomarkers of response in subjects with FH. Seventeen adult FH patients underwent a 6-week washout followed by 4-week treatment with atorvastatin (80 mg/day) or rosuvastatin (40 mg/day). LDL-c response was considered good (40-70 % reduction, n = 9) or poor (3-33 % reduction, n = 8). Serum lipidomic profile was analyzed by ultra-high-performance liquid chromatography combined with electrospray ionization tandem time-of-flight mass spectrometry, and data were analyzed using MetaboAnalyst v5.0. Lipidomic analysis identified 353 lipids grouped into 16 classes. Statin treatment reduced drastically 8 of 13 lipid classes, generating a characteristic lipidomic profile with a significant contribution of phosphatidylinositols (PI) 16:0/18:2, 18:0/18:1 and 18:0/18:2; and triacylglycerols (TAG) 18:2x2/18:3, 18:1/18:2/18:3, 16:1/18:2x2, 16:1/18:2/18:3 and 16:1/18:2/Arachidonic acid (p-adjusted <0.05). Biomarker analysis implemented in MetaboAnalyst subsequently identified PI 16:1/18:0, 16:0/18:2 and 18:0/18:2 as predictors of statin response with and receiver operating characteristic (ROC) areas under the curve of 0.98, 0.94 and 0.91, respectively. In conclusion, statins extensively modulate the overall serum lipid composition of FH individuals and these findings suggest that phosphatidyl-inositol molecules are potential predictive biomarkers of statin response.

Influence of polymorphisms in IRS1, IRS2, MC3R, and MC4R on metabolic and inflammatory status and food intake in Brazilian adults: An exploratory pilot study.

Polymorphisms in genes of leptin-melanocortin and insulin pathways have been associated with obesity and type 2 diabetes. We hypothesized that polymorphisms in IRS1, IRS2, MC3R, and MC4R influence metabolic and inflammatory markers and food intake composition in Brazilian subjects. This exploratory pilot study included 358 adult subjects. Clinical, anthropometric, and laboratory data were obtained through interview and access to medical records. The variants IRS1 rs2943634 A˃C, IRS2 rs1865434 C>T, MC3R rs3746619 C>A, and MC4R rs17782313 T>C were analyzed by real-time polymerase chain reaction. Food intake composition was assessed in a group of subjects with obesity (n = 84) before and after a short-term nutritional counseling program (9 weeks). MC4R rs17782313 was associated with increased risk of obesity (P = .034). Multivariate linear regression analysis adjusted by covariates indicated associations of IRS2 rs1865434 with reduced low-density lipoprotein cholesterol and resistin, MC3R rs3746619 with high glycated hemoglobin, and IRS1 rs2943634 and MC4R rs17782313 with increased high-sensitivity C-reactive protein (P < .05). Energy intake and carbohydrate and total fat intakes were reduced after the diet-oriented program (P < .05). Multivariate linear regression analysis showed associations of IRS2 rs1865434 with high basal fiber intake, IRS1 rs2943634 with low postprogram carbohydrate intake, and MC4R rs17782313 with low postprogram total fat and saturated fatty acid intakes (P < .05). Although significant associations did not survive correction for multiple comparisons using the Benjamini-Hochberg method in this exploratory study, polymorphisms in IRS1, IRS2, MC3R, and MC4R influence metabolic and inflammatory status in Brazilian adults. IRS1 and MC4R variants may influence carbohydrate, total fat, and saturated fatty acid intakes in response to a diet-oriented program in subjects with obesity.

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

BACKGROUND: 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. METHODS AND RESULTS: 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). CONCLUSION: LDLR and PCSK9 3´UTR variants disturb miRNA:mRNA interactions that could affect gene expression and are potentially associated with molecular diagnosis of FH.

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

BACKGROUND 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. METHODS AND RESULTS 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). CONCLUSION LDLR and PCSK9 3´UTR variants disturb miRNA:mRNA interactions that could affect gene expression and are potentially associated with molecular diagnosis of FH.

Identification of apob variants in samples from the brazilian population with hypercholesterolemic phenotype

INTRODUCTION AND OBJECTIVES: Apolipoprotein B plays a crucial role in regulating plasma cholesterol by mediating the interaction of low-density lipoprotein (LDL) with LDL receptors in the liver. Inherited mutations in this gene may increase the risk of developing premature atherosclerotic cardiovascular disease, especially in individuals with familial hypercholesterolemia type 2 (FH2). The aim of this study is to identify APOB variants that may indicate pathogenicity in a sample of the Brazilian population using a data bank exome sequencing study by NGS in a Brazilian population phenotypically diagnosed by clinical and laboratory profile. This finding is going to improve genetic hypercholesteremia diagnosis. Casuistic, Material and METHODS: High quality DNA samples (n=300) were sequenced using an exon- targeted gene sequencing (ETGS) strategy to identify variants in FH-related genes. Pathogenicity classification was based on criteria established by the American College of Medical Genetics and Genomics (ACMG), also using information from ClinVar and pathogenicity scores from previous association studies. RESULTS and CONCLUSIONS: A total of 121 variants were identified in APOB, of which four are novel variants missense (p.Thr626Asn, p.Ile2750Thr, p.Gln2078Lys and p.Met4184Arg). After curating pathogenicity scores, variants were classified according to the ACMG criteria. Among them four as pathogenic or likely pathogenic (p.Pro2739Leu, p.His1923Arg, p.Pro994Leu and p.Pro877Leu), and 21 variants had uncertain significance. Additionally, 92 previously known variants with uncertain significance were classified as benign or likely benign. The results were submitted to Clinvar for actualization of pathogenicity and to improve the molecular diagnosis associating APOB variants with the clinical phenotype of hypercholesterolemia. Financing: FAPESP, CNPQ, CAPES.

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.

The let-7b-5p, miR-326, and miR-125a-3p are associated with left ventricular systolic dysfunction in post-myocardial infarction.

Background: Acute ST-elevation myocardial infarction (STEMI) can lead to adverse cardiac remodeling, resulting in left ventricular systolic dysfunction (LVSd) and heart failure. Epigenetic regulators, such as microRNAs, may be involved in the physiopathology of LVSd. Objective: This study explored microRNAs in peripheral blood mononuclear cells (PBMC) of post-myocardial infarction patients with LVSd. Methods: Post-STEMI patients were grouped as having (LVSd, n = 9) or not LVSd (non-LVSd, n = 16). The expression of 61 microRNAs was analyzed in PBMC by RT-qPCR and the differentially expressed microRNAs were identified. Principal Component Analysis stratified the microRNAs based on the development of dysfunction. Predictive variables of LVSd were investigated through logistic regression analysis. A system biology approach was used to explore the regulatory molecular network of the disease and an enrichment analysis was performed. Results: The let-7b-5p (AUC: 0.807; 95% CI: 0.63-0.98; p = 0.013), miR-125a-3p (AUC: 0.800; 95% CI: 0.61-0.99; p = 0.036) and miR-326 (AUC: 0.783; 95% CI: 0.54-1.00; p = 0.028) were upregulated in LVSd (p < 0.05) and discriminated LVSd from non-LVSd. Multivariate logistic regression analysis showed let-7b-5p (OR: 16.00; 95% CI: 1.54-166.05; p = 0.020) and miR-326 (OR: 28.00; 95% CI: 2.42-323.70; p = 0.008) as predictors of LVSd. The enrichment analysis revealed association of the targets of these three microRNAs with immunological response, cell-cell adhesion, and cardiac changes. Conclusion: LVSd alters the expression of let-7b-5p, miR-326, and miR-125a-3p in PBMC from post-STEMI, indicating their potential involvement in the cardiac dysfunction physiopathology and highlighting these miRNAs as possible LVSd biomarkers.

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.

In silico analysis of upstream variants in Brazilian patients with familial hypercholesterolemia

ABSTRACT: Familial hypercholesterolemia (FH) is a prevalent autosomal genetic disease associated with increased risk of early cardiovascular events and death due to chronic exposure to very high levels of low-density lipoprotein cholesterol (LDL-c). Pathogenic variants in the coding regions of LDLR, APOB and PCSK9 account for most FH cases, and variants in non-coding regions maybe involved in FH as well. Variants in the upstream region of LDLR, APOB and PCSK9 were screened by targeted next-generation sequencing and their effects were explored using in silico tools. Twenty-five patients without pathogenic variants in FH-related genes were selected. 3 kb upstream regions of LDLR, APOB and PCSK9 were sequenced using the AmpliSeq (Illumina) and Miseq Reagent Nano Kit v2 (Illumina). Sequencing data were analyzed using variant discovery and functional annotation tools. Potentially regulatory variants were selected by integrating data from public databases, published data and context-dependent regulatory prediction score. Thirty-four single nucleotide variants (SNVs) in upstream regions were identified (6 in LDLR, 15 in APOB, and 13 in PCSK9). Five SNVs were prioritized as potentially regulatory variants (rs934197, rs9282606, rs36218923, rs538300761, g.55038486A > G). APOB rs934197 was previously associated with increased rate of transcription, which in silico analysis suggests that could be due to reducing binding affinity of a transcriptional repressor. Our findings highlight the importance of variant screening outside of coding regions of all relevant genes. Further functional studies are necessary to confirm that prioritized variants could impact gene regulation and contribute to the FH phenotype.

Effects of PCSK9 missense variants on molecular conformation and biological activity in transfected HEK293FT cells.

PCSK9 gain-of-function (GOF) variants increase degradation of low-density lipoprotein receptor (LDLR) and are potentially associated with Familial Hypercholesterolemia (FH). This study aimed to explore the effects of PCSK9 missense variants on protein structure and interactions with LDLR using molecular modeling analyses and in vitro functional studies. Variants in FH-related genes were identified in a Brazilian FH cohort using an exon-target gene sequencing strategy. Eight PCSK9 missense variants in pro- [p.(E32K) and p.(E57K)], catalytic [p.(R237W), p.(P279T) and p.(A443T)], and C-terminal histidine-cysteine rich (CHR) [p.(R469W), p.(Q619P) and p.(R680Q)] domains were identified. Molecular dynamics analyses revealed that GOF variants p.(E32K) and p.(R469W) increased extreme motions in PCSK9 amino acid backbone fluctuations and affected Hbond and water bridge interactions between the pro-domain and CM1 region of the CHR domain. HEK293FT cells transfected with plasmids carrying p.(E32K) and p.(R469W) variants reduced LDLR expression (8.7 % and 14.8 %, respectively) compared to wild type (p < 0.05) but these GOF variants did not affect PCSK9 expression and secretion. The missense variants p.(P279T) and p.(Q619P) also reduced protein stability and altered Hbond interactions. In conclusion, PCSK9 p.(E32K), p.(R469W), p.(P279T) and p.(Q619P) variants disrupt intramolecular interactions that are essential for PCSK9 structural conformation and biological activity and may have a potential role in FH pathogenesis.

In silico analysis of upstream variants in Brazilian patients with Familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is a prevalent autosomal genetic disease associated with increased risk of early cardiovascular events and death due to chronic exposure to very high levels of low-density lipoprotein cholesterol (LDL-c). Pathogenic variants in the coding regions of LDLR, APOB and PCSK9 account for most FH cases, and variants in non-coding regions maybe involved in FH as well. Variants in the upstream region of LDLR, APOB and PCSK9 were screened by targeted next-generation sequencing and their effects were explored using in silico tools. Twenty-five patients without pathogenic variants in FH-related genes were selected. 3 kb upstream regions of LDLR, APOB and PCSK9 were sequenced using the AmpliSeq (Illumina) and Miseq Reagent Nano Kit v2 (Illumina). Sequencing data were analyzed using variant discovery and functional annotation tools. Potentially regulatory variants were selected by integrating data from public databases, published data and context-dependent regulatory prediction score. Thirty-four single nucleotide variants (SNVs) in upstream regions were identified (6 in LDLR, 15 in APOB, and 13 in PCSK9). Five SNVs were prioritized as potentially regulatory variants (rs934197, rs9282606, rs36218923, rs538300761, g.55038486A > G). APOB rs934197 was previously associated with increased rate of transcription, which in silico analysis suggests that could be due to reducing binding affinity of a transcriptional repressor. Our findings highlight the importance of variant screening outside of coding regions of all relevant genes. Further functional studies are necessary to confirm that prioritized variants could impact gene regulation and contribute to the FH phenotype.

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.

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

ABSTRACT: 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.

TREML4 polymorphisms increase the mRNA in blood leukocytes in the progression of atherosclerosis.

TREML4 and other members of the triggering receptor expressed in the myeloid cell family are associated with a risk of atherosclerosis and progression in coronary artery disease, acute coronary syndrome, and coronary artery calcification. Herein, the relationship between TREML4 expression and its polymorphisms (rs2803495 and rs280396) was evaluated in patients with subclinical atherosclerosis (n = 340) and heart failure post-acute myocardial infarction (MI) (n = 68) for the first time. TREML4 variants rs2803495 (A > G) and rs2803496 (T > C) and leukocyte mRNA expression was analyzed by qRT-PCR. The rs2803495 G allele was associated with TREML4 expression (OR 8.01, CI 3.78-16.99, p < 0.001). Patients carrying the rs2803496 C minor allele (TC/CC genotypes) were more likely to express TREML4 than those without the C allele (OR 10.42, CI 4.76-22.78, p < 0.001), as well as having higher levels of TREML4 expression (OR 4.88, CI 2.35-10.12, p < 0.001). Thus, we report for the first time that TREML4 is not associated with the early stages of atherosclerotic plaque formation and later stages after MI. In conclusion, TREML4 mRNA expression in blood leukocytes is influenced by minor alleles (G and C) and may regulate differently during the atherosclerosis progression stages, but not in asymptomatic atherosclerosis disease and post-MI.

Effects of PCSK9 missense variants on molecular conformation and biological activity in transfected HEK293FT cells

ABSTRACT: PCSK9 gain-of-function (GOF) variants increase degradation of low-density lipoprotein receptor (LDLR) and are potentially associated with Familial Hypercholesterolemia (FH). This study aimed to explore the effects of PCSK9 missense variants on protein structure and interactions with LDLR using molecular modeling analyses and in vitro functional studies. Variants in FH-related genes were identified in a Brazilian FH cohort using an exon-target gene sequencing strategy. Eight PCSK9 missense variants in pro- [p.(E32K) and p.(E57K)], catalytic [p.(R237W), p.(P279T) and p.(A443T)], and C-terminal histidine-cysteine rich (CHR) [p.(R469W), p.(Q619P) and p.(R680Q)] domains were identified. Molecular dynamics analyses revealed that GOF variants p.(E32K) and p.(R469W) increased extreme motions in PCSK9 amino acid backbone fluctuations and affected Hbond and water bridge interactions between the pro-domain and CM1 region of the CHR domain. HEK293FT cells transfected with plasmids carrying p.(E32K) and p.(R469W) variants reduced LDLR expression (8.7 % and 14.8 %, respectively) compared to wild type (p < 0.05) but these GOF variants did not affect PCSK9 expression and secretion. The missense variants p.(P279T) and p.(Q619P) also reduced protein stability and altered Hbond interactions. In conclusion, PCSK9 p.(E32K), p.(R469W), p.(P279T) and p.(Q619P) variants disrupt intramolecular interactions that are essential for PCSK9 structural conformation and biological activity and may have a potential role in FH pathogenesis.

TREML4 polymorphisms increase the mRNA in blood leukocytes in the progression of atherosclerosis

ABSTRACT: TREML4 and other members of the triggering receptor expressed in the myeloid cell family are associated with a risk of atherosclerosis and progression in coronary artery disease, acute coronary syndrome, and coronary artery calcification. Herein, the relationship between TREML4 expression and its polymorphisms (rs2803495 and rs280396) was evaluated in patients with subclinical atherosclerosis (n = 340) and heart failure post-acute myocardial infarction (MI) (n = 68) for the first time. TREML4 variants rs2803495 (A > G) and rs2803496 (T > C) and leukocyte mRNA expression was analyzed by qRT-PCR. The rs2803495 G allele was associated with TREML4 expression (OR 8.01, CI 3.78-16.99, p < 0.001). Patients carrying the rs2803496 C minor allele (TC/CC genotypes) were more likely to express TREML4 than those without the C allele (OR 10.42, CI 4.76-22.78, p < 0.001), as well as having higher levels of TREML4 expression (OR 4.88, CI 2.35-10.12, p < 0.001). Thus, we report for the first time that TREML4 is not associated with the early stages of atherosclerotic plaque formation and later stages after MI. In conclusion, TREML4 mRNA expression in blood leukocytes is influenced by minor alleles (G and C) and may regulate differently during the atherosclerosis progression stages, but not in asymptomatic atherosclerosis disease and post-MI.

Influence of antidepressant drugs on DNA methylation of ion channels genes in blood cells of psychiatric patients

ABSTRACT AIM: This study investigated the influence of antidepressant drugs on methylation status of KCNE1, KCNH2 and SCN5A promoters and ECG parameters in adult psychiatric patients. MATERIALS & METHODS: Electrocardiographic evaluation (24 h) and blood samples were obtained from 34 psychiatric patients before and after 30 days of antidepressant therapy. Methylation of promoter CpG sites of KCNE1, KCNH2 and SCN5A was analyzed by pyrosequencing. RESULTS: Three CpG and four CpG sites of KCNE1 and SCN5A, respectively, had increased % methylation after treatment. Principal component analysis showed correlations of the methylation status with electrocardiographic variables, antidepressant doses and patient age. CONCLUSION: Short-term treatment with antidepressant drugs increase DNA methylation in KCNE1 and SCN5A promoters, which may induce ECG alterations in psychiatric patients.