Predicted deleterious variants in ABCA1, LPL, LPA and KIF6 are associated with statin response and adverse events in patients with familial hypercholesterolemia and disturb protein structure and stability

OBJECTIVES: This study explored the association of deleterious variants in pharmacodynamics (PD) genes with statin response and adverse effects in patients with familial hypercholesterolemia (FH) and analyzed their potential effects on protein structure and stability. METHODS: Clinical and laboratory data were obtained from 144 adult FH patients treated with statins. A panel of 32 PD genes was analyzed by exon-targeted gene sequencing. Deleterious variants were identified using prediction algorithms and their structural effects were analyzed by molecular modeling studies. RESULTS: A total of 102 variants were predicted as deleterious (83 missense, 8 stop-gain, 4 frameshift, 1 indel, 6 splicing). The variants ABCA1 rs769705621 (indel), LPA rs41267807 (p.Tyr2023Cys) and KIF6 rs20455 (p.Trp719Arg) were associated with reduced low-density lipoprotein cholesterol (LDLc) response to statins, and the LPL rs1801177 (p.Asp36Asn) with increased LDLc response (P < 0.05). LPA rs3124784 (p.Arg2016Cys) was predicted to increase statin response (P = 0.022), and ABCA1 rs769705621 to increase the risk of statin-related adverse events (SRAE) (P = 0.027). LPA p.Arg2016Cys and LPL p.Asn36Asp maintained interactions with solvent, LPA p.Tyr2023Cys reduced intramolecular interaction with Gln1987, and KIF6 p.Trp719Arg did not affect intramolecular interactions. DDMut analysis showed that LPA p.Arg2016Cys and p.Tyr2023Cys and LPL p.Asp36Asn caused energetically favorable changes, and KIF6 p.Trp719Arg resulted in unfavorable energetic changes, affecting protein stability. CONCLUSION: Deleterious variants in ABCA1, LPA, LPL and KIF6 are associated with variability in LDLc response to statins, and ABCA1 rs769705621 is associated with SRAE risk in FH patients. Molecular modeling studies suggest that LPA p.Tyr2023Cys and KIF6 p.Trp719Arg disturb protein conformational structure and stability.

Predicted deleterious variants in ABCA1, LPL, LPA and KIF6 are associated with statin response and adverse events in patients with familial hypercholesterolemia and disturb protein structure and stability.

OBJECTIVES: This study explored the association of deleterious variants in pharmacodynamics (PD) genes with statin response and adverse effects in patients with familial hypercholesterolemia (FH) and analyzed their potential effects on protein structure and stability. METHODS: Clinical and laboratory data were obtained from 144 adult FH patients treated with statins. A panel of 32 PD genes was analyzed by exon-targeted gene sequencing. Deleterious variants were identified using prediction algorithms and their structural effects were analyzed by molecular modeling studies. RESULTS: A total of 102 variants were predicted as deleterious (83 missense, 8 stop-gain, 4 frameshift, 1 indel, 6 splicing). The variants ABCA1 rs769705621 (indel), LPA rs41267807 (p.Tyr2023Cys) and KIF6 rs20455 (p.Trp719Arg) were associated with reduced low-density lipoprotein cholesterol (LDLc) response to statins, and the LPL rs1801177 (p.Asp36Asn) with increased LDLc response (P < 0.05). LPA rs3124784 (p.Arg2016Cys) was predicted to increase statin response (P = 0.022), and ABCA1 rs769705621 to increase the risk of statin-related adverse events (SRAE) (P = 0.027). LPA p.Arg2016Cys and LPL p.Asn36Asp maintained interactions with solvent, LPA p.Tyr2023Cys reduced intramolecular interaction with Gln1987, and KIF6 p.Trp719Arg did not affect intramolecular interactions. DDMut analysis showed that LPA p.Arg2016Cys and p.Tyr2023Cys and LPL p.Asp36Asn caused energetically favorable changes, and KIF6 p.Trp719Arg resulted in unfavorable energetic changes, affecting protein stability. CONCLUSION: Deleterious variants in ABCA1, LPA, LPL and KIF6 are associated with variability in LDLc response to statins, and ABCA1 rs769705621 is associated with SRAE risk in FH patients. Molecular modeling studies suggest that LPA p.Tyr2023Cys and KIF6 p.Trp719Arg disturb protein conformational structure and stability.

Single-nucleotide polymorphisms related to vitamin D metabolism and severity or mortality of COVID-19: A systematic review and meta-analysis.

This systematic review and meta-analysis aimed to verify the association between single-nucleotide polymorphisms (SNPs) in vitamin D-related genes and the severity or mortality of coronavirus disease 19 (COVID-19). We systematically searched PubMed, BVS/Bireme, Scopus, Embase, and Web of Science for relevant studies published until November 24, 2023. Twelve studies were included. Thirty-one SNPs related to four genes were studied (VDR, 13 SNPs; GC, 6 SNPs; DHCR7/NADSYN1, 6 SNPs; CYP2R1, 6 SNPs). Eight SNPs were examined in two or more studies (VDR rs731236, rs2228570, rs1544410, rs7975232, rs739837, rs757343, rs11568820, and rs4516035). Meta-analysis showed a significant association between the VDR rs1544410 Bb + bb genotype and b allele and an increased odds of developing severe/critical COVID-19 (Bb + bb vs. BB = 2 studies, OR = 1.73, 95% confidence interval (CI): 1.16-2.57, P = 0.007, I2 = 0%; b allele vs. B allele = 2 studies, OR = 1.31, 95% CI: 1.03-1.67; P = 0.03; I2 = 0%). Regarding the mortality rate, VDR rs731236 TT-genotype, TT + Tt genotype, and T allele; VDR rs1544410 bb-genotype, Bb + bb genotype, and b allele; VDR rs7975232 AA-genotype, AA + Aa genotype, and A allele; and VDR rs2228570 ff-genotype, Ff + ff genotype, and f allele were associated with increased odds of death due to COVID-19. In conclusion, the present study suggests that SNPs rs1544410 may serve as a predictive biomarker for COVID-19 severity and rs731236, rs1544410, rs7975232, and rs2228570 as predictive biomarkers for COVID-19 mortality. More well-designed studies involving a larger number of COVID-19 patients are required to validate and replicate these findings.

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.

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.

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 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 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.

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.

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

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.

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.

Genetic Variant ABCC1 rs45511401 Is Associated with Increased Response to Statins in Patients with Familial Hypercholesterolemia.

Statins are the first-line treatment for familial hypercholesterolemia (FH), but response is highly variable due to genetic and nongenetic factors. Here, we explored the association between response and genetic variability in 114 Brazilian adult FH patients. Specifically, a panel of 84 genes was analyzed by exon-targeted gene sequencing (ETGS), and the functional impact of variants in pharmacokinetic (PK) genes was assessed using an array of functionality prediction methods. Low-density lipoprotein cholesterol (LDL-c) response to statins (reduction ≥ 50%) and statin-related adverse event (SRAE) risk were assessed in carriers of deleterious variants in PK-related genes using multivariate linear regression analyses. Fifty-eight (50.8%) FH patients responded to statins, and 24 (21.0%) had SRAE. Results of the multivariate regression analysis revealed that ABCC1 rs45511401 significantly increased LDL-c reduction after statin treatment (p < 0.05). In silico analysis of the amino-acid change using molecular docking showed that ABCC1 rs45511401 possibly impairs statin efflux. Deleterious variants in PK genes were not associated with an increased risk of SRAE. In conclusion, the deleterious variant ABCC1 rs45511401 enhanced LDL-c response in Brazilian FH patients. As such, this variant might be a promising candidate for the individualization of statin therapy.

Genetic variant ABCC1 rs45511401 is associated with increased response to statins in patients with familial hypercholesterolemia

Statins are the first-line treatment for familial hypercholesterolemia (FH), but response is highly variable due to genetic and nongenetic factors. Here, we explored the association between response and genetic variability in 114 Brazilian adult FH patients. Specifically, a panel of 84 genes was analyzed by exon-targeted gene sequencing (ETGS), and the functional impact of variants in pharmacokinetic (PK) genes was assessed using an array of functionality prediction methods. Low-density lipoprotein cholesterol (LDL-c) response to statins (reduction ≥ 50%) and statin-related adverse event (SRAE) risk were assessed in carriers of deleterious variants in PK-related genes using multivariate linear regression analyses. Fifty-eight (50.8%) FH patients responded to statins, and 24 (21.0%) had SRAE. Results of the multivariate regression analysis revealed that ABCC1 rs45511401 significantly increased LDL-c reduction after statin treatment (p < 0.05). In silico analysis of the amino-acid change using molecular docking showed that ABCC1 rs45511401 possibly impairs statin efflux. Deleterious variants in PK genes were not associated with an increased risk of SRAE. In conclusion, the deleterious variant ABCC1 rs45511401 enhanced LDL-c response in Brazilian FH patients. As such, this variant might be a promising candidate for the individualization of statin therapy.

Pharmacogenomics of statins: lipid response and other outcomes in Brazilian cohorts.

Statins are inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme in cholesterol biosynthesis, that are highly effective in reducing plasma low-density lipoprotein (LDL) cholesterol and decreasing the risk of cardiovascular events. In recent years, a multitude of variants in genes involved in pharmacokinetics (PK) and pharmacodynamics (PD) have been suggested to influence the cholesterol-lowering response. However, the vast majority of studies have analyzed the pharmacogenetic associations in populations in Europe and the USA, whereas data in other populations, including Brazil, are mostly lacking. This narrative review provides an update of clinical studies on statin pharmacogenomics in Brazilian cohorts exploring lipid-lowering response, adverse events and pleiotropic effects. We find that variants in drug transporter genes (SLCO1B1 and ABCB1) positively impacted atorvastatin and simvastatin response, whereas variants in genes of drug metabolizing enzymes (CYP3A5) decreased response. Furthermore, multiple associations of variants in PD genes (HMGCR, LDLR and APOB) with statin response were identified. Few studies have explored statin-related adverse events, and only ABCB1 but not SLCO1B1 variants were robustly associated with increased risk in Brazil. Statin-related pleiotropic effects were shown to be influenced by variants in PD (LDLR, NR1H2) and antioxidant enzyme (NOS3, SOD2, MTHFR, SELENOP) genes. The findings of these studies indicate that statin pharmacogenomic associations are distinctly different in Brazil compared to other populations. This review also discusses the clinical implications of pharmacogenetic studies and the rising importance of investigating rare variants to explore their association with statin response.

Pharmacogenomics of Antihypertensive Drugs in Brazil: Recent Progress and Clinical Implications.

BACKGROUND: The available antihypertensive drugs are effective and well tolerated agents. However, only about half of patients with treated hypertension achieve appropriate blood pressure control. Genetic and non-genetic factors contribute to the interindividual variability of the therapeutic response. OBJECTIVE: This review constitutes a comprehensive update of the pharmacogenomics of antihypertensive drugs and their clinical implications in Brazil. RESULTS: Twenty-five studies explored the influence of gene variants on drug response in patients with primary, resistant, or gestational hypertension. Variants in BDKRB2, NOS3, PRKCA, and VEGFA influenced the response to enalapril in patients with primary hypertension. AGT and MMP2 variants were associated with a high risk of resistance to antihypertensive treatment, whereas NOS2 variants were related to low risk. Moreover, NAT2 slow acetylators showed an increased response to hydralazine in patients with resistant hypertension. HMOX1, NAMPT, MMP9, NOS3, and TIMP1 variants might be markers of drug responsiveness in hypertensive or preeclamptic pregnant women. Power and replication of studies, polygenic nature of the response to therapy, and treatment with multiple drugs were important challenges to identify genetic predictors of antihypertensive response in Brazil. CONCLUSION: Pharmacogenomic studies in Brazilian cohorts provide some evidence of variants, mainly in pharmacodynamics genes, which influence the response to antihypertensive drugs. However, some findings are limited by cohort size or therapeutic scheme and may be influenced by interactions with other genetic and non-genetic factors. Therefore, further investigations are needed to elucidate the contribution of pharmacogenomics to the efficacy and safety of antihypertensive therapy.

Cardiovascular Pharmacogenomics: An Update on Clinical Studies of Antithrombotic Drugs in Brazilian Patients.

Anticoagulant and antiplatelet drugs effectively prevent thrombotic events in patients with cardiovascular diseases, ischemic stroke, peripheral vascular diseases, and other thromboembolic diseases. However, genetic and non-genetic factors affect the response to antithrombotic therapy and can increase the risk of adverse events. This narrative review discusses pharmacogenomic studies on antithrombotic drugs commonly prescribed in Brazil. Multiple Brazilian studies assessed the impact of pharmacokinetic (PK) and pharmacodynamic (PD) gene variants on warfarin response. The reduced function alleles CYP2C9*2 and CYP2C9*3, and VKORC1 rs9923231 (c.-1639G>A) are associated with increased sensitivity to warfarin and a low dose requirement to prevent bleeding episodes, whereas CYP4F2 rs2108622 (p.Val433Met) carriers have higher dose requirements (warfarin resistance). These deleterious variants and non-genetic factors (age, gender, body weight, co-administered drugs, food interactions, and others) account for up to 63% of the warfarin dose variability. Few pharmacogenomics studies have explored antiplatelet drugs in Brazilian cohorts, finding associations between CYP2C19*2, PON1 rs662 and ABCC3 rs757421 genotypes and platelet responsiveness or clopidogrel PK in subjects with coronary artery disease (CAD) or acute coronary syndrome (ACS), whereas ITGB3 contributes to aspirin PK but not platelet responsiveness in diabetic patients. Brazilian guidelines on anticoagulants and antiplatelets recommend the use of a platelet aggregation test or genotyping only in selected cases of ACS subjects without ST-segment elevation taking clopidogrel, and also suggest CYP2C9 and VKORC1 genotyping before starting warfarin therapy to assess the risk of bleeding episodes or warfarin resistance.