Identification of the proprotein convertase subtilisin/kexin type 9 (PCSK9) as the third gene cau... more Identification of the proprotein convertase subtilisin/kexin type 9 (PCSK9) as the third gene causing familial hypercholesterolemia (FH) and understanding its complex biology has led to the discovery of a novel class of therapeutic agents. PCSK9 undergoes autocatalytic cleavage in the endoplasmic reticulum and enters the secretory pathway. The PCSK9 gene is under the regulatory control of sterol receptor binding proteins 1 and 2. Statins increase PCSK9 and this may modulate the response to this class of medications. In plasma, PCSK9 binds to the epidermal growth factor-like domain of the LDL receptor (LDL-R) on the cell and, once incorporated in the late endosomal pathway, directs the LDL-R toward lysosomal degradation rather than recycling to the plasma membrane. Thus, gain-of-function PCSK9 mutations lead to an FH phenotype, whereas loss-of-function mutations are associated with increased LDL-R-mediated endocytosis of LDL particles and lower LDL cholesterol in plasma. Inhibition o...
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of circulating low densi... more Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of circulating low density lipoprotein cholesterol (LDL-C) levels. Besides its full-length mature form, multiple variants of PCSK9 have been reported such as forms that are truncated, mutated and/or with posttranslational modifications (PTMs). Previous studies have demonstrated that most of these variants affect PCSK9's function and thereby LDL-C levels. Commercial ELISA kits are available for quantification of PCSK9, but do not allow discrimination between the various forms and PTMs of the protein. To address this issue and given the complexity and wide dynamic range of the plasma proteome, we have developed a mass spectrometric immunoassay coupled to selected reaction monitoring (MSIA-SRM) for the multiplexed quantification of several forms of circulating PCSK9 in human plasma. Our MSIA-SRM assay quantifies peptides spanning the various protein domains and the S688 phosphorylation site. The assay was applied in two distinct cohorts of obese patients and healthy pregnant women stratified by their circulating LDL-C levels. Seven PCSK9 peptides were monitored in plasma samples: one in the prodomain prior to the autocleavage site at Q152, one in the catalytic domain prior to the furin cleavage site at R218, two in the catalytic domain following R218, one in the cysteine and histidine rich domain (CHRD) and the C-terminal peptide phosphorylated at S688 and unmodified. The latter was not detectable in sufficient amounts to be quantified in human plasma. All peptides were measured with high reproducibility and with LLOQ and LOD below the clinical range. The abundance of 5 of the 6 detectable PCSK9 peptides was higher in obese patients stratified with high circulating LDL-C levels as compared to those with low LDL-C (p<0.05). The same 5 peptides showed good and statistically significant correlations with LDL-C levels (0.55<r<0.65; 0.0002⩽p⩽0.002), but not the S688 phosphorylated peptide. However, this phosphopeptide was significantly correlated with insulin resistance (r=0.48; p=0.04). In the pregnant women cohort, none of the peptides were associated to LDL-C levels. However, the 6 detectable PCSK9 peptides, but not PCSK9 measured by ELISA, were significantly correlated with serum triglyceride levels in this cohort. Our results also suggest that PCSK9 circulates with S688 phosphorylated at high stoichiometry. In summary, we have developed and applied a robust and sensitive MSIA-SRM assay for the absolute quantification of all PCSK9 domains and a PTM in human plasma. This assay revealed novel relationships between PCSK9 and metabolic phenotypes, as compared to classical ELISA assays.
Low levels of high-density lipoprotein (HDL) cholesterol constitutes a major risk factor for athe... more Low levels of high-density lipoprotein (HDL) cholesterol constitutes a major risk factor for atherosclerosis. Recent studies from our group reported a genetic association between the WW domain-containing oxidoreductase (WWOX) gene and HDL cholesterol levels. Here, through next-generation resequencing, in vivo functional studies and gene microarray analyses, we investigated the role of WWOX in HDL and lipid metabolism. Using next-generation resequencing of the WWOX region, we first identified 8 variants significantly associated and perfectly segregating with the low-HDL trait in 2 multigenerational French Canadian dyslipidemic families. To understand in vivo functions of WWOX, we used liver-specific Wwox(hep-/-) and total Wwox(-/-) mice models, where we found decreased ApoA-I and Abca1 levels in hepatic tissues. Analyses of lipoprotein profiles in Wwox(-/-), but not Wwox(hep-/-) littermates, also showed marked reductions in serum HDL cholesterol concentrations, concordant with the lo...
Homocysteine is an amino acid that is toxic to vascular endothelial cells, and plasma elevations ... more Homocysteine is an amino acid that is toxic to vascular endothelial cells, and plasma elevations have been associated with venous thromboembolism. Severe hyperhomocysteinemia (>100 μmol/L) may result from mutations in the genes coding for enzymes in the trans-sulfuration or the folate/vitamin B12-dependent re-methylation pathways. Here, we report the case of a young woman with severe, recurrent thrombo-embolic events associated with severe hyperhomocysteinemia (111 μmol/L). We identified a homozygous mutation in the cystathionine β -synthase gene (p.I278T) and the presence of the Factor V Leiden mutation. Family study shows segregation of elevated homocysteine in heterozygous relatives for the mutation in the cystathionine β -synthase gene. Management consisted of anticoagulation with warfarin and supplementation with folate, vitamin B6 (pyridoxine) and vitamin B12. After twelve years of follow-up, plasma homocysteine levels remain in the moderate range (~20 μmol/L, reference ran...
Heart disease and stroke represent the major burden of health worldwide and account for a stagger... more Heart disease and stroke represent the major burden of health worldwide and account for a staggering 17 million deaths yearly. This pandemic is, in great part preventable through simple and modifiable preventive measures such as smoking cessation, healthy eating, regular activity and weight loss. In patients with established atherosclerotic vascular disease, lipid lowering agent have had a major impact on reducing risk, along with pharmacological treatment of elevated blood pressure and the use of anti-thrombotic medication. Despite these advances, there remains a significant residual risk and newer approaches are required to decrease atherosclerosis. Innate and acquired immunity play a pivotal role in the initiation, progression and instability of the atherosclerotic plaque. The remarkable complexity of the immune system makes it difficult to target a single pathway for the prevention of cardiovascular disease. Nevertheless, recent data points to possible therapeutic targets that may decrease atherosclerosis, without increasing the risk of infection, decreasing immune surveillance for cancers and without undue toxicity. Here we discuss the clinical trials and registry data associated with the use of inflammation modulation and cardiovascular disease and the ongoing major clinical trial that may change the clinical medicine and preventive cardiology. The selective inhibition of interleukin 1β and the use of low-dose methotrexate are now undergoing large outcome-driven clinical trials to answer these questions.
Clinical Approach to Sudden Cardiac Death Syndromes, 2009
... Genetic Lipoprotein Disorders and Cardiovascular Disease Khalid Alwaili, Khalid Alrasadi, Zar... more ... Genetic Lipoprotein Disorders and Cardiovascular Disease Khalid Alwaili, Khalid Alrasadi, Zari Dastani, Iulia Iatan, Zuhier Awan, and Jacques Genest ... Most of the cholesterol in plasma circulates in the form of cho-lesteryl esters, in the core of lipoprotein particles. ...
Familial hypercholesterolemia (FH) due to mutations in the low-density lipoprotein receptor (LDLR... more Familial hypercholesterolemia (FH) due to mutations in the low-density lipoprotein receptor (LDLR) gene exhibit severe, premature aortic calcification in a gene-dosage, age-dependent fashion. We sought to determine potential associations with mineral and skeletal indices. We obtained computed tomography (CT) scan aortic calcium scores (AoCSs) in 19 (age 49 [SD 14] years) FH patients heterozygous for the 15-kb deletion at the LDLR gene and examined associations with various indices of mineral and skeletal homeostasis. We found that mean bone mineral density (BMD) at the femoral neck in these patients did not differ from age-, sex-, and province-matched mean BMD, and we observed no association of AoCS with any marker of bone resorption. However, there were negative correlations between AoCS and serum concentrations of osteocalcin, a marker of bone formation (r = -0.64, P = 0.0034), urinary calcium (r = -0.59, P = 0.0085), and estimated glomerular filtration rate (r = -0.67, P = 0.0019). We found that LDLR-deficient FH was not associated with obvious bone loss or a major disturbance in calcium homeostasis. The lack of LDLR, however, may modify osteoblast function or extracellular calcium distribution, manifesting as lower bone formation, and reduced calcium excretion, resulting in increased deposition in calcifying vascular tissue.
PCSK9 (proprotein convertase subtilisin/kexin type 9) promotes the degradation of the LDLR (LDL r... more PCSK9 (proprotein convertase subtilisin/kexin type 9) promotes the degradation of the LDLR (LDL receptor) in hepatocytes, leading to an increase in plasma LDL-C (LDL cholesterol). Previous animal studies have shown that insulin stimulates PCSK9 transcription and observational human studies showed a positive correlation between plasma PCSK9 concentration and fasting insulinemia. The purpose of this study was to investigate the effects of chronic and acute hyperinsulinemia on PCSK9 in a large cohort of human subjects as well as at a cellular level. The in vivo effect of hyperinsulinemia on plasma PCSK9 concentration was studied using euglycemic-hyperinsulinemic clamps in 82 non-diabetic post-menopausal obese patients. We studied the in vitro effects of insulin stimulation on PCSK9 mRNA as well as on protein expression and secretion in HepG2 and Huh7 cells. Analysis of the pre and post-clamp data revealed a 15.4% (p<0.001) lowering of plasma PCSK9 concentration after acute insulin induction. In vitro studies post-insulin stimulation showed that mRNA levels of PCSK9 reduced by 25% in HepG2 cells (p<0.027) and by 59% in Huh7 cells (p<0.01). Intracellular concentration of PCSK9 were 10% lower in HepG2 cells (p<0.05) and 35% lower in Huh7 cells (p<0.05). Our results show an inhibitory effect of acute hyperinsulinemia on PCSK9 in humans both in vitro and in vivo. This data may assist in evaluating PCSK9 levels in individuals on insulin therapy.
Once thought to be a passive process of calcium accrual in arterial vascular beds, vascular calci... more Once thought to be a passive process of calcium accrual in arterial vascular beds, vascular calcification is considered to be a tightly regulated process mediated by osteoblast-like cells under the dys-regulated interplay of shear stress, metabolites, cytokines and transcription factors. Unfortunately, without effective medical interventions to prevent or regress vascular calcification, this process directly contributes to cardiovascular morbidity and mortality. We have previously shown that patients with familial hypercholesterolemia (FH) have severe, premature aortic calcification and calcific aortic stenosis. We showed an age-related gene-dosage effect of deletion of the low-density lipoprotein receptor (LDL-R) gene on aortic calcification in human subjects with FH. The LDL-R deficient mouse and transgenic mice overexpressing the LDL-R degrading protein PCSK9 also exhibit aortic calcification, not fully explained by increased LDL cholesterol levels. Taken together, these data suggests a novel role for the LDL-R in the inhibition of vascular calcification. Understanding the molecular role of the LDL-R and its signaling partners in vascular calcification will be instrumental in identifying novel therapies for a common age-related process associated with a large burden of disease.
Natural loss-of-function mutations in the proprotein convertase subtilisin/kexin type-9 gene (PCS... more Natural loss-of-function mutations in the proprotein convertase subtilisin/kexin type-9 gene (PCSK9) are associated with lower cholesterol and cardiovascular risk. Because a founder effect exists in French Canadians for many lipid-related genes, we sought to investigate PCSK9 mutations and associated variables in this population. We also investigated the combined effect of PCSK9 mutations and the apolipoprotein E (apoE) polymorphism on metabolic variables. Gene sequencing and screening was carried out in 1745 healthy individuals ages 9, 13, and 16 years from a provincially representative population sample. In parallel, we measured related metabolic markers and used appropriate statistical methods. We report herein that the carrier rates of the R46L single-nucleotide polymorphism were higher in the French Canadian population (4.8%) than previously seen in Caucasian individuals (2.4%). This is second to the most common variant, insertion of leucine, at a carrier rate of 24%, making it the most common PCSK9 loss-of-function mutation in French Canadian individuals. In R46L carriers, the contribution of the apoE genotype better explains the cholesterol phenotype than the R46L mutation alone. Patients, with both the R46L and apoE3/E2 genotype also showed a tendency toward insulin resistance as indicated by a 2-fold increase in insulin, homeostasis model assessment of insulin resistance, and leptin concentrations, compared with those without apoE3/E2. R46L and insertion of leucine mutations were more frequent in French Canadian individuals and showed a specific geographic distribution. This might represent a gene selection to overcome clustering genes harbouring familial hypercholesterolemia and might suggest a founder effect. Subjects with the apoE3/E2 genotype and R46L have increased plasma insulin, homeostasis model assessment of insulin resistance, and leptin, an intriguing finding that warrants further investigation.
Identification of the proprotein convertase subtilisin/kexin type 9 (PCSK9) as the third gene cau... more Identification of the proprotein convertase subtilisin/kexin type 9 (PCSK9) as the third gene causing familial hypercholesterolemia (FH) and understanding its complex biology has led to the discovery of a novel class of therapeutic agents. PCSK9 undergoes autocatalytic cleavage in the endoplasmic reticulum and enters the secretory pathway. The PCSK9 gene is under the regulatory control of sterol receptor binding proteins 1 and 2. Statins increase PCSK9 and this may modulate the response to this class of medications. In plasma, PCSK9 binds to the epidermal growth factor-like domain of the LDL receptor (LDL-R) on the cell and, once incorporated in the late endosomal pathway, directs the LDL-R toward lysosomal degradation rather than recycling to the plasma membrane. Thus, gain-of-function PCSK9 mutations lead to an FH phenotype, whereas loss-of-function mutations are associated with increased LDL-R-mediated endocytosis of LDL particles and lower LDL cholesterol in plasma. Inhibition o...
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of circulating low densi... more Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of circulating low density lipoprotein cholesterol (LDL-C) levels. Besides its full-length mature form, multiple variants of PCSK9 have been reported such as forms that are truncated, mutated and/or with posttranslational modifications (PTMs). Previous studies have demonstrated that most of these variants affect PCSK9's function and thereby LDL-C levels. Commercial ELISA kits are available for quantification of PCSK9, but do not allow discrimination between the various forms and PTMs of the protein. To address this issue and given the complexity and wide dynamic range of the plasma proteome, we have developed a mass spectrometric immunoassay coupled to selected reaction monitoring (MSIA-SRM) for the multiplexed quantification of several forms of circulating PCSK9 in human plasma. Our MSIA-SRM assay quantifies peptides spanning the various protein domains and the S688 phosphorylation site. The assay was applied in two distinct cohorts of obese patients and healthy pregnant women stratified by their circulating LDL-C levels. Seven PCSK9 peptides were monitored in plasma samples: one in the prodomain prior to the autocleavage site at Q152, one in the catalytic domain prior to the furin cleavage site at R218, two in the catalytic domain following R218, one in the cysteine and histidine rich domain (CHRD) and the C-terminal peptide phosphorylated at S688 and unmodified. The latter was not detectable in sufficient amounts to be quantified in human plasma. All peptides were measured with high reproducibility and with LLOQ and LOD below the clinical range. The abundance of 5 of the 6 detectable PCSK9 peptides was higher in obese patients stratified with high circulating LDL-C levels as compared to those with low LDL-C (p<0.05). The same 5 peptides showed good and statistically significant correlations with LDL-C levels (0.55<r<0.65; 0.0002⩽p⩽0.002), but not the S688 phosphorylated peptide. However, this phosphopeptide was significantly correlated with insulin resistance (r=0.48; p=0.04). In the pregnant women cohort, none of the peptides were associated to LDL-C levels. However, the 6 detectable PCSK9 peptides, but not PCSK9 measured by ELISA, were significantly correlated with serum triglyceride levels in this cohort. Our results also suggest that PCSK9 circulates with S688 phosphorylated at high stoichiometry. In summary, we have developed and applied a robust and sensitive MSIA-SRM assay for the absolute quantification of all PCSK9 domains and a PTM in human plasma. This assay revealed novel relationships between PCSK9 and metabolic phenotypes, as compared to classical ELISA assays.
Low levels of high-density lipoprotein (HDL) cholesterol constitutes a major risk factor for athe... more Low levels of high-density lipoprotein (HDL) cholesterol constitutes a major risk factor for atherosclerosis. Recent studies from our group reported a genetic association between the WW domain-containing oxidoreductase (WWOX) gene and HDL cholesterol levels. Here, through next-generation resequencing, in vivo functional studies and gene microarray analyses, we investigated the role of WWOX in HDL and lipid metabolism. Using next-generation resequencing of the WWOX region, we first identified 8 variants significantly associated and perfectly segregating with the low-HDL trait in 2 multigenerational French Canadian dyslipidemic families. To understand in vivo functions of WWOX, we used liver-specific Wwox(hep-/-) and total Wwox(-/-) mice models, where we found decreased ApoA-I and Abca1 levels in hepatic tissues. Analyses of lipoprotein profiles in Wwox(-/-), but not Wwox(hep-/-) littermates, also showed marked reductions in serum HDL cholesterol concentrations, concordant with the lo...
Homocysteine is an amino acid that is toxic to vascular endothelial cells, and plasma elevations ... more Homocysteine is an amino acid that is toxic to vascular endothelial cells, and plasma elevations have been associated with venous thromboembolism. Severe hyperhomocysteinemia (>100 μmol/L) may result from mutations in the genes coding for enzymes in the trans-sulfuration or the folate/vitamin B12-dependent re-methylation pathways. Here, we report the case of a young woman with severe, recurrent thrombo-embolic events associated with severe hyperhomocysteinemia (111 μmol/L). We identified a homozygous mutation in the cystathionine β -synthase gene (p.I278T) and the presence of the Factor V Leiden mutation. Family study shows segregation of elevated homocysteine in heterozygous relatives for the mutation in the cystathionine β -synthase gene. Management consisted of anticoagulation with warfarin and supplementation with folate, vitamin B6 (pyridoxine) and vitamin B12. After twelve years of follow-up, plasma homocysteine levels remain in the moderate range (~20 μmol/L, reference ran...
Heart disease and stroke represent the major burden of health worldwide and account for a stagger... more Heart disease and stroke represent the major burden of health worldwide and account for a staggering 17 million deaths yearly. This pandemic is, in great part preventable through simple and modifiable preventive measures such as smoking cessation, healthy eating, regular activity and weight loss. In patients with established atherosclerotic vascular disease, lipid lowering agent have had a major impact on reducing risk, along with pharmacological treatment of elevated blood pressure and the use of anti-thrombotic medication. Despite these advances, there remains a significant residual risk and newer approaches are required to decrease atherosclerosis. Innate and acquired immunity play a pivotal role in the initiation, progression and instability of the atherosclerotic plaque. The remarkable complexity of the immune system makes it difficult to target a single pathway for the prevention of cardiovascular disease. Nevertheless, recent data points to possible therapeutic targets that may decrease atherosclerosis, without increasing the risk of infection, decreasing immune surveillance for cancers and without undue toxicity. Here we discuss the clinical trials and registry data associated with the use of inflammation modulation and cardiovascular disease and the ongoing major clinical trial that may change the clinical medicine and preventive cardiology. The selective inhibition of interleukin 1β and the use of low-dose methotrexate are now undergoing large outcome-driven clinical trials to answer these questions.
Clinical Approach to Sudden Cardiac Death Syndromes, 2009
... Genetic Lipoprotein Disorders and Cardiovascular Disease Khalid Alwaili, Khalid Alrasadi, Zar... more ... Genetic Lipoprotein Disorders and Cardiovascular Disease Khalid Alwaili, Khalid Alrasadi, Zari Dastani, Iulia Iatan, Zuhier Awan, and Jacques Genest ... Most of the cholesterol in plasma circulates in the form of cho-lesteryl esters, in the core of lipoprotein particles. ...
Familial hypercholesterolemia (FH) due to mutations in the low-density lipoprotein receptor (LDLR... more Familial hypercholesterolemia (FH) due to mutations in the low-density lipoprotein receptor (LDLR) gene exhibit severe, premature aortic calcification in a gene-dosage, age-dependent fashion. We sought to determine potential associations with mineral and skeletal indices. We obtained computed tomography (CT) scan aortic calcium scores (AoCSs) in 19 (age 49 [SD 14] years) FH patients heterozygous for the 15-kb deletion at the LDLR gene and examined associations with various indices of mineral and skeletal homeostasis. We found that mean bone mineral density (BMD) at the femoral neck in these patients did not differ from age-, sex-, and province-matched mean BMD, and we observed no association of AoCS with any marker of bone resorption. However, there were negative correlations between AoCS and serum concentrations of osteocalcin, a marker of bone formation (r = -0.64, P = 0.0034), urinary calcium (r = -0.59, P = 0.0085), and estimated glomerular filtration rate (r = -0.67, P = 0.0019). We found that LDLR-deficient FH was not associated with obvious bone loss or a major disturbance in calcium homeostasis. The lack of LDLR, however, may modify osteoblast function or extracellular calcium distribution, manifesting as lower bone formation, and reduced calcium excretion, resulting in increased deposition in calcifying vascular tissue.
PCSK9 (proprotein convertase subtilisin/kexin type 9) promotes the degradation of the LDLR (LDL r... more PCSK9 (proprotein convertase subtilisin/kexin type 9) promotes the degradation of the LDLR (LDL receptor) in hepatocytes, leading to an increase in plasma LDL-C (LDL cholesterol). Previous animal studies have shown that insulin stimulates PCSK9 transcription and observational human studies showed a positive correlation between plasma PCSK9 concentration and fasting insulinemia. The purpose of this study was to investigate the effects of chronic and acute hyperinsulinemia on PCSK9 in a large cohort of human subjects as well as at a cellular level. The in vivo effect of hyperinsulinemia on plasma PCSK9 concentration was studied using euglycemic-hyperinsulinemic clamps in 82 non-diabetic post-menopausal obese patients. We studied the in vitro effects of insulin stimulation on PCSK9 mRNA as well as on protein expression and secretion in HepG2 and Huh7 cells. Analysis of the pre and post-clamp data revealed a 15.4% (p<0.001) lowering of plasma PCSK9 concentration after acute insulin induction. In vitro studies post-insulin stimulation showed that mRNA levels of PCSK9 reduced by 25% in HepG2 cells (p<0.027) and by 59% in Huh7 cells (p<0.01). Intracellular concentration of PCSK9 were 10% lower in HepG2 cells (p<0.05) and 35% lower in Huh7 cells (p<0.05). Our results show an inhibitory effect of acute hyperinsulinemia on PCSK9 in humans both in vitro and in vivo. This data may assist in evaluating PCSK9 levels in individuals on insulin therapy.
Once thought to be a passive process of calcium accrual in arterial vascular beds, vascular calci... more Once thought to be a passive process of calcium accrual in arterial vascular beds, vascular calcification is considered to be a tightly regulated process mediated by osteoblast-like cells under the dys-regulated interplay of shear stress, metabolites, cytokines and transcription factors. Unfortunately, without effective medical interventions to prevent or regress vascular calcification, this process directly contributes to cardiovascular morbidity and mortality. We have previously shown that patients with familial hypercholesterolemia (FH) have severe, premature aortic calcification and calcific aortic stenosis. We showed an age-related gene-dosage effect of deletion of the low-density lipoprotein receptor (LDL-R) gene on aortic calcification in human subjects with FH. The LDL-R deficient mouse and transgenic mice overexpressing the LDL-R degrading protein PCSK9 also exhibit aortic calcification, not fully explained by increased LDL cholesterol levels. Taken together, these data suggests a novel role for the LDL-R in the inhibition of vascular calcification. Understanding the molecular role of the LDL-R and its signaling partners in vascular calcification will be instrumental in identifying novel therapies for a common age-related process associated with a large burden of disease.
Natural loss-of-function mutations in the proprotein convertase subtilisin/kexin type-9 gene (PCS... more Natural loss-of-function mutations in the proprotein convertase subtilisin/kexin type-9 gene (PCSK9) are associated with lower cholesterol and cardiovascular risk. Because a founder effect exists in French Canadians for many lipid-related genes, we sought to investigate PCSK9 mutations and associated variables in this population. We also investigated the combined effect of PCSK9 mutations and the apolipoprotein E (apoE) polymorphism on metabolic variables. Gene sequencing and screening was carried out in 1745 healthy individuals ages 9, 13, and 16 years from a provincially representative population sample. In parallel, we measured related metabolic markers and used appropriate statistical methods. We report herein that the carrier rates of the R46L single-nucleotide polymorphism were higher in the French Canadian population (4.8%) than previously seen in Caucasian individuals (2.4%). This is second to the most common variant, insertion of leucine, at a carrier rate of 24%, making it the most common PCSK9 loss-of-function mutation in French Canadian individuals. In R46L carriers, the contribution of the apoE genotype better explains the cholesterol phenotype than the R46L mutation alone. Patients, with both the R46L and apoE3/E2 genotype also showed a tendency toward insulin resistance as indicated by a 2-fold increase in insulin, homeostasis model assessment of insulin resistance, and leptin concentrations, compared with those without apoE3/E2. R46L and insertion of leucine mutations were more frequent in French Canadian individuals and showed a specific geographic distribution. This might represent a gene selection to overcome clustering genes harbouring familial hypercholesterolemia and might suggest a founder effect. Subjects with the apoE3/E2 genotype and R46L have increased plasma insulin, homeostasis model assessment of insulin resistance, and leptin, an intriguing finding that warrants further investigation.
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