Aortic valve and arterial calcification in patients with familial hypercholesterolemia.

Heterozygous familial hypercholesterolemia (heFH) is an autosomal dominant lipid metabolism disorder. Its prevalence is 1:250-1:300 people in the population. Patients with heFH have an up to 13-fold increased risk of premature coronary artery disease (CAD). If left untreated, men and women with heFH typically develop early CAD before the ages of 55 and 60, respectively. There is evidence that coronary artery calcification (CAC) and aortic valve calcification (AoVC) are more prevalent in FH patients than in the general population. It is documented that CAC and AoVC are predictors of increased risk of cardiovascular morbidity and mortality in heFH patients, like in the general population. However, the etiology and pathogenesis of vascular calcification in FH patients is not well understood. Risk factors for vascular calcification include age, increased levels of atherogenic lipoproteins, Lp(a), increased blood pressure, and inflammation. There are convincing data from clinical studies and animal atherosclerotic mouse models using low-density lipoprotein receptor (LDL-R) knockout mice that the vascular calcification processes in FH are associated with LDL-R mutations, probably partly due to a higher total cholesterol burden of FH subjects. Data from animal models as well as clinical studies indicate that the Wnt/beta-catenin pathway components and LDL receptor-related proteins 5 and 6 (LRP-5/6) might be involved in calcification processes in FH patients. The purpose of the review is to describe the prevalence of coronary and aortic calcification and its risk factors in FH patients. The review covers data about the role of the Wnt/beta-catenin pathway and factors modulating calcification processes.

Targeted sequencing of a gene panel in patients with familial hypercholesterolemia from Southern Poland.

INTRODUCTION: Familial hypercholesterolemia (FH) is an autosomal dominant monogenic lipid metabolism disorder characterized by a significantly elevated level of low­density lipoprotein (LDL) cholesterol and leading to premature ischemic heart disease. FH is caused by mutations in the LDLR, APOB, and PCSK9 genes; however, these mutations account for only about 40% of FH cases. In order to obtain a genetic diagnosis of FH, sequencing of other genes involved in the lipid metabolism might be useful. OBJECTIVES: This study aimed to describe genetic variants in genes associated with FH in a group of patients from the Malopolska province in Southern Poland, using the targeted next generation sequencing (NGS) technology. PATIENTS AND METHODS: The study involved 90 unrelated adults (age range, 18-70 years) with FH diagnosed clinically according to the Simon Broome Register criteria. A custom­designed capture assay and the Illumina MiSeq platform were used. The panel included exons and exon / intron boundaries of known FH­causing genes: LDLR, APOB, and PCSK9, as well as genes previously associated with high cholesterol levels: APOE, ABCG5, ABCG8, LPL, NPC1, LDLRAP1, LIPC, STAP1, and CELSR2. Genetic variants were classified based on in silico predictions and ClinVar reports. RESULTS: We detected 4 patients with variants in the LDLR and APOB genes that had not been previously linked to FH in ClinVar. We also found APOB mutations outside the common LDL receptor-binding region, in exons 26 and 29. Interestingly, we observed a high frequency of pathogenic variants in exon 4 of the APOE gene: rs7412, probably damaging (4 patients) and rs429358, benign (16 patients). CONCLUSIONS: NGS is a useful and reliable method to detect new variants in genes related to FH. In addition, the results enable the detection of FH phenocopies and introduction of appropriate treatment.

Effectiveness and safety of PCSK9 inhibitor therapy in patients with familial hypercholesterolemia within a therapeutic program in Poland: Preliminary multicenter data.

BACKGROUND: In Poland, treatment with proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors has become available free of charge in a therapeutic program. Assessed herein, is the efficacy and safety of alirocumab and evolocumab in patients with heterozygous familial hypercholesterolemia (FH). METHODS: Data of 55 adult FH patients who participated in the program were analyzed upon meeting the criteria established by the Ministry of Health (low density lipoprotein cholesterol [LDL-C] above 160 mg/dL on max. tolerated statin dose and ezetimib). The efficacy of PCSK9 inhibitors in reducing LDL-C with drug administration every 2 weeks was assessed after 3 months and 1 year of therapy. A safety profile evaluation was performed at each visit. 48 patients completed the 3-month and 21 for the 1-year observation periods (34 patients treated with alirokumab and 14 with evolocumab). RESULTS: The mean concentration of direct-measured LDL-C decreased from the initial level of 215.1 ± 74.5 mg/dL to 75.3 ± 64.1 mg/dL, i.e., by 65 ± 14% following 3 months of treatment. This effect was stable in 1-year observation (77.7 ± 72.8 mg/dL). Adverse effects were flu-like symptoms (13.0%), injection site reactions (11.1%), fatigue (5.6%) and musculoskeletal symptoms (5.6%). Seven patients failed to complete the 3-month treatment period due to side effects or non-compliance, and 1 patient failed to complete the 1-year treatment due to myalgia. CONCLUSIONS: This study confirmed high effectiveness of PCSK9 inhibitors in reducing LDL-C levels in patients with FH. Due to restrictive inclusion criteria with LDL-C threshold level > 160 mg/dL (> 4.1 mmol/L) required for participation in the therapeutic program, a relatively small number of FH patients were eligible for treatment.

Serum PCSK9 Correlates with PTX3 and Apolipoproteins B, A1, and C3 Concentrations in Patients with Type 2 Diabetes.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is involved in the regulation of LDL metabolism. There is evidence that circulating PCSK9 is a cardiovascular risk factor. In this study, we determined factors associated with circulating PCSK9 in a group of patients with type 2 diabetes mellitus (DM2). Material included 116 consecutive patients with DM2 from outpatient diabetes clinic. Circulating PCSK9, PTX3, apolipoprotein (apo) B100, apo B48, and apo C3 levels were determined by ELISA, apo A1 by immunoturbidimetry. The mean (sd) age of patients was 59.1 (11.1) years, the mean (sd) values of serum PCSK9 were 255.4 (106.97) ng/ml. Circulating PCSK9 correlated negatively with age (r = -0.21, p < 0.05) and HbA1c (r = -0.21, p < 0.05) and positively with BMI (r = 0.21, p < 0.05), total cholesterol (r = 0.59), LDL-cholesterol (r = 0.50), triglyceride (r = 0.35), apo B100 (r = 0.43), apo A1 (r = 0.43) (p < 0.001 for all), apo C3 (r = 0.29, p < 0.01), and apo B48 (r = 0.25, p < 0.01) concentration and FLI (r = 0.26, p < 0.01). Strong correlation between PTX3 and PCSK9 levels was observed (r = 0.47, p < 0.001). Multiple stepwise backward regression analysis with PCSK9 as dependent variable revealed that PTX3, apo B100, apo A1, apo B48, and BMI were significantly positive and the presence of NAFLD and HbA1c negatively associated with PCSK9 concentrations. These variables together explain 57% of PCSK9 variability; the strongest relationship was observed between PCSK9 and PTX3 and apo B100. Our results indicate that circulating PCSK9 is significantly associated with inflammation marker PTX3 as well as atherogenic lipids and apolipoproteins C3, B100, and B48, which might be of value in understanding interactions between development of atherosclerosis and inflammatory state in DM2 patients.

Correlates of pentraxin 3 serum concentration in men and women with type 2 diabetes.

Elevated levels of plasma pentraxin 3 (PTX3), a marker of inflammation, are associated with the risk of developing cardiovascular diseases in the general population, as well as in patients with type 2 diabetes (DM2). In this study, we aimed to determine factors associated with PTX3 serum concentrations in men and women with DM2. The study included 116 consecutive patients (67 men and 49 women) with DM2 from an outpatient diabetic clinic. Men were characterised by lower age and higher uric acid, creatinine and bilirubin concentrations and waist/hip ratio than women. In women, low-density lipoprotein cholesterol (LDL-C) levels were higher than in men. In men, median (interquartile range) values of PTX3 concentration were 4.02 (1.99), and in women they were 4.53 (3.31) ng/ml (NS). In men, PTX3 concentrations correlated with total cholesterol (TC), triglycerides, apolipoprotein (Apo) C3, Apo B48, Glc and creatinine levels. In women, PTX3 correlated significantly with TC and LDL-C and Apo B100. Partial regression analysis revealed that after adjusting for age, PTX3 concentrations in men were significantly associated with TC, LDL-C, triglycerides, creatinine, Apo C3 and Apo B48, while in women they were associated with TC, LDL-C and Apo B100. The results could be of importance in sex-specific prevention of vascular complications in DM2 patients.

Serum pentraxin 3 concentration in patients with type 2 diabetes and nonalcoholic fatty liver disease.

INTRODUCTION: Nonalcoholic fatty liver disease (NAFLD) is common in patients with type 2 diabetes (T2D). Pentraxin 3 (PTX3), a marker of inflammation, is a cardiovascular risk factor. OBJECTIVES: We examined clinical and biochemical factors associated with serum PTX3 concentrations in patients with T2D with and without NAFLD. PATIENTS AND METHODS: Serum material was obtained from 116 patients with T2D (mean age, 59.1 years), including 79 patients with NAFLD. RESULTS: Median (interquartile range) PTX3 level was 4.264 (2.293) ng/ml in patients with and 3.773 (3.223) ng/ml in patients without NAFLD (P = 0.93). In the whole group, PTX3 level was associated with total cholesterol, low­density lipoprotein cholesterol (LDL­C), apolipoprotein (apo) B100, apo C3, triglyceride (TG) concentrations, and waist circumference after adjustment for age and gender. As indicated by partial regression coefficient b, increase of independent variable LDL­C by 1 mmol/l was associated with the rise of PTX3 by 1.2017 ng/ml, increase of apo B100 by 1 mg/dl with the rise of PTX3 by 1.0051 ng/ml, and increase of apo C3 by 1 µg/dl with the rise of PTX3 by 1.0012 ng/ml. In patients with T2D with NAFLD, total cholesterol, LDL­C, TG, apo C3, and apo B100 were associated with PTX3. Associations of PTX3 with apolipoproteins were observed only in the NAFLD group. CONCLUSIONS: Reported associations of PTX3 level add new insight into possible mechanisms of its atherogenic actions.

Carotid artery plaques - Are risk factors the same in men and women with familial hypercholesterolemia?

BACKGROUND AND AIMS: High low-density lipoprotein (LDL)-cholesterol levels are a major cause of premature coronary heart disease (CHD) and death in patients with familial hypercholesterolemia (FH). It is uncertain whether these risk factors affect men and women equally. We aimed to compare the risk factors of carotid plaques, which are reliable surrogates of coronary atherosclerosis, in men and women with FH. METHODS: 154 patients with FH (40.9% men) were included, diagnosed according to Simon Broome criteria. Carotid plaques were assessed by ultrasound. RESULTS: In women multiple logistic regression analysis revealed that systolic blood pressure, high-density lipoprotein-cholesterol (HDL-C), apolipoprotein (apo) A1, and alanine aminotransferase (ALT) were associated with the presence of carotid plaques. In this female cohort, the age adjusted odds ratio for the increase of HDL-C by 1 standard deviation was related to a 55% decrease in the odds of having carotid plaques (p=0.01) and the age adjusted odds ratio for the increase of ALT by 1U/L was related to a 7% in the increase odds of having carotid plaques (p=0.02). In men, in multiple logistic regression analysis only apo B concentration was significantly related to carotid plaque presence. The odds ratio for the increase of apo B by 1mg/dl corresponded to a 4% increase in the odds of having carotid plaques (p=0.01) and, interestingly, in men not treated with statin, this ratio reached 8% (p=0.04). CONCLUSIONS: In summary, our study suggests a difference in risk factors of carotid artery plaques between men and women with FH.

The genetic spectrum of familial hypercholesterolemia in south-eastern Poland.

BACKGROUND: Familial hypercholesterolemia (FH) is a common autosomal dominant disorder with a frequency of 1 in 200 to 500 in most European populations. Mutations in LDLR, APOB and PCSK9 genes are known to cause FH. In this study, we analyzed the genetic spectrum of the disease in the understudied Polish population. MATERIALS AND METHODS: 161 unrelated subjects with a clinical diagnosis of FH from the south-eastern region of Poland were recruited. High resolution melt and direct sequencing of PCR products were used to screen 18 exons of LDLR, a region of exon 26 in the APOB gene and exon 7 of PCSK9. Multiplex ligation-dependent probe amplification (MLPA) was performed to detect gross deletions and insertions in LDLR. Genotypes of six LDL-C raising SNPs were used for a polygenic gene score calculation. RESULTS: We found 39 different pathogenic mutations in the LDLR gene with 10 of them being novel. 13 (8%) individuals carried the p.Arg3527Gln mutation in APOB, and overall the detection rate was 43.4%. Of the patients where no mutation could be found, 53 (84.1%) had a gene score in the top three quartiles of the healthy comparison group suggesting that they have a polygenic cause for their high cholesterol. CONCLUSIONS: These results confirm the genetic heterogeneity of FH in Poland, which should be considered when designing a diagnostic strategy in the country. As in the UK, in the majority of patients where no mutation can be found, there is likely to be a polygenic cause of their high cholesterol level.

Carotid Plaques Correlates in Patients With Familial Hypercholesterolemia.

Patients with familial hypercholesterolemia (FH) are at increased risk of premature cardiovascular disease. We compared factors associated with the presence of carotid plaques and carotid intima-media thickness (cIMT), markers of subclinical atherosclerosis, in 241 patients with FH (98, 40.7% men; mean age 41 ± 18.4 years). Patients with FH having carotid plaques (36.5%) had mean age, apolipoprotein (apo) B, glucose, apoA1, systolic blood pressure (SBP) and diastolic BP, waist/hip ratio (WHR), and body mass index higher than patients without plaques. Logistic regression revealed that apoB (odds ratio [OR] per 1 unit change 1.03,P= .005), high-density lipoprotein cholesterol (HDL-C; OR per 1 standard deviation [SD] change 0.59,P= .015), and non-HDL-C (OR per 1SD change 1.53,P= .04) were significantly associated with the presence of plaques. The cIMT correlated with obesity parameters, BP, apoB, glucose, high-sensitivity C-reactive protein, creatinine, γ-glutamyl transpeptidase, and alanine transaminase (P< .001). Regression analysis revealed that cIMT was significantly associated with apoB, SBP, and WHR. These results confirm the role of apoB-containing lipoproteins and low HDL-C with the presence of carotid plaques and apoB, BP, and WHR with cIMT.

Refinement of variant selection for the LDL cholesterol genetic risk score in the diagnosis of the polygenic form of clinical familial hypercholesterolemia and replication in samples from 6 countries.

BACKGROUND: Familial hypercholesterolemia (FH) is an autosomal-dominant disorder caused by mutations in 1 of 3 genes. In the 60% of patients who are mutation negative, we have recently shown that the clinical phenotype can be associated with an accumulation of common small-effect LDL cholesterol (LDL-C)-raising alleles by use of a 12-single nucleotide polymorphism (12-SNP) score. The aims of the study were to improve the selection of SNPs and replicate the results in additional samples. METHODS: We used ROC curves to determine the optimum number of LDL-C SNPs. For replication analysis, we genotyped patients with a clinical diagnosis of FH from 6 countries for 6 LDL-C-associated alleles. We compared the weighted SNP score among patients with no confirmed mutation (FH/M-), those with a mutation (FH/M+), and controls from a UK population sample (WHII). RESULTS: Increasing the number of SNPs to 33 did not improve the ability of the score to discriminate between FH/M- and controls, whereas sequential removal of SNPs with smaller effects/lower frequency showed that a weighted score of 6 SNPs performed as well as the 12-SNP score. Metaanalysis of the weighted 6-SNP score, on the basis of polymorphisms in CELSR2 (cadherin, EGF LAG 7-pass G-type receptor 2), APOB (apolipoprotein B), ABCG5/8 [ATP-binding cassette, sub-family G (WHITE), member 5/8], LDLR (low density lipoprotein receptor), and APOE (apolipoprotein E) loci, in the independent FH/M- cohorts showed a consistently higher score in comparison to the WHII population (P < 2.2 × 10(-16)). Modeling in individuals with a 6-SNP score in the top three-fourths of the score distribution indicated a >95% likelihood of a polygenic explanation of their increased LDL-C. CONCLUSIONS: A 6-SNP LDL-C score consistently distinguishes FH/M- patients from healthy individuals. The hypercholesterolemia in 88% of mutation-negative patients is likely to have a polygenic basis.

GTP cyclohydrolase I gene polymorphisms are associated with endothelial dysfunction and oxidative stress in patients with type 2 diabetes mellitus.

BACKGROUND: The genetic background of atherosclerosis in type 2 diabetes mellitus (T2DM) is complex and poorly understood. Studying genetic components of intermediate phenotypes, such as endothelial dysfunction and oxidative stress, may aid in identifying novel genetic components for atherosclerosis in diabetic patients. METHODS: Five polymorphisms forming two haplotype blocks within the GTP cyclohydrolase 1 gene, encoding a rate limiting enzyme in tetrahydrobiopterin synthesis, were studied in the context of flow and nitroglycerin mediated dilation (FMD and NMD), intima-media thickness (IMT), and plasma concentrations of von Willebrand factor (vWF) and malondialdehyde (MDA). RESULTS: Rs841 was associated with FMD (p = 0.01), while polymorphisms Rs10483639, Rs841, Rs3783641 (which form a single haplotype) were associated with both MDA (p = 0.012, p = 0.0015 and p = 0.003, respectively) and vWF concentrations (p = 0.016, p = 0.03 and p = 0.045, respectively). In addition, polymorphism Rs8007267 was also associated with MDA (p = 0.006). Haplotype analysis confirmed the association of both haplotypes with studied variables. CONCLUSIONS: Genetic variation of the GCH1 gene is associated with endothelial dysfunction and oxidative stress in T2DM patients.

[Determinants of nonalcoholic fatty liver disease in men and women with type 2 diabetes]. / Determinanty niealkoholowego stluszczenia watroby u mezczyzn i kobiet z cukrzyca typu 2.

Nonalcoholic fatty liver disease (NAFLD) is recognized in 20-30% of general population but among the people with impaired glucose metabolism this percentage is about 70-90%. The aim of this study is to assess the determinants of NAFLD with respect to patients' gender. We examined 180 patients, 73 women and 107 men. Increased body mass index (BMI), waist circumference above the norm are the determinants of NAFLD irrespectively to gender. Besides it has been observed that in the women group the increase of HDL-cholesterol by 1 mmol/l decreases the chances of NAFLD occurrence by 90%.

Nonalcoholic fatty liver disease is associated with low HDL cholesterol and coronary angioplasty in patients with type 2 diabetes.

BACKGROUND: There is evidence that nonalcoholic fatty liver disease (NAFLD) is associated with increased cardiovascular risk. In this study we examined factors associated with the presence of NAFLD and the prevalence of macroangiopathy in patients with type 2 diabetes. MATERIAL AND METHODS: Subjects were 101 consecutive patients with type 2 diabetes: 72 with NAFLD and 29 free of NAFLD. NAFLD was diagnosed by ultrasonography. Serum lipids were measured enzymatically and glycated hemoglobin HbA1c was measured by HPLC. RESULTS: The mean age of patients was 53.1 ± 10.4 in the NAFLD group and 44.9 ± 10.9 years in patients without NAFLD (p<0.001). The mean duration of diabetes was 10 ± 6.3 years in patients with NAFLD and 15.1 ± 7.8 years in those without NAFLD (p<0.001). Mean values of glycated hemoglobin A1c were similar in both groups. Patients with NAFLD were characterized by a significantly higher prevalence of coronary angioplasty (20.8% vs. 0%, p=0.008). Overweight and obesity were observed in a higher percentage of patients with NAFLD (p<0.001). Patients with NAFLD were characterized by significantly higher values of alanine transaminase (p=0.033), and lower serum concentrations of HDL-cholesterol (p<0.001) and creatinine (p=0.034). Logistic regression analysis (p<0.001) revealed that NAFLD was positively associated with waist circumference above normal (women >80 cm, men >94 cm) (p=0.0083) and alanine transaminase activity (p=0.0164), and negatively with creatinine concentration (p=0.0226). In a second logistic regression model (p<0.001), waist circumference (p<0.007) and total cholesterol (p<0.008) were positive predictors, while HDL-C (p<0.003) was a negative predictor of NAFLD. CONCLUSIONS: The results of the study suggest that NAFLD is associated with visceral obesity and low HDL-cholesterol in patients with type 2 diabetes.

Intima-media thickness correlates with features of metabolic syndrome in young people with a clinical diagnosis of familial hypercholesterolaemia.

BACKGROUND: Familial hypercholesterolaemia (FH) is a monogenic lipid metabolism disorder characterised by markedly elevated serum low-density lipoprotein (LDL) cholesterol level due to a mutation in the LDL receptor gene. Clinical features of FH include premature atherosclerosis and coronary artery disease. AIM: To explore associations between noninvasive markers of atherosclerosis including intima-media thickness (IMT) and pulse wave velocity (PWV) and blood lipids, blood pressure (BP) and obesity in a group of young patients with FH. METHODS: Study population included 36 patients aged < 35 years with the diagnosis of FH based on the Simon Broome Register criteria, and their 49 relatives who comprised the control group free of FH. RESULTS: Mean IMT values were higher in FH patients than controls (0.60 ± 0.19 vs. 0.53 ± 0.07 mm, respectively, p < 0.05).Mean body mass index (BMI) and waist circumference were similar in patients and controls. The prevalence of carotid atherosclerotic plaques was significantly higher among FH patients (n = 6) than in controls (n = 1) (21.4% vs. 2.6%, p = 0.012). Arterial hypertension was present in 27.8% of patients with FH and 16.3% of subjects in the control group. Systolic blood pressure (SBP) in FH patients correlated significantly with age (r = 0.35), BMI (r = 0.48) and waist circumference (r = 0.47), and diastolic blood pressure (DBP) correlated with BMI (r = 0.42) and waist circumference (r = 0.41). PWV correlated significantly with age (r = 0.44), SBP (r = 0.63) and DBP (r = 0.52). We did not find any correlations between IMT and serum lipids, BP or obesity indices in FH patients. CONCLUSIONS: Our findings show a higher rate of arterial hypertension in young FH patients compared to their relatives free of FH, with significant associations between BP and indices of obesity and arterial stiffness. Intensive lipid lowering and antihypertensive therapy along with a reduction in central fat may be considered a mandatory treatment strategy in young FH patients to prevent atherosclerosis and increased arterial stiffness.

A novel mutation (Cys308Phe) of the LDL receptor gene in families from the South-Eastern part of Poland.

The purpose of this investigation was to characterize a new mutation in the LDL-receptor (LDLR) gene in three families with clinically diagnosed familial hypercholesterolemia (FH) from the South-Eastern part of Poland. Mutational screening with exon by exon sequencing analysis was performed in all probands. The novel mutation c986G>T (Cys308Phe) in the exon 7 of LDLR gene was found in three apparently unrelated probands with FH. Analysis of the receptor activity of peripheral blood lymphocytes by binding and uptake of DiL-LDL showed a significant reduction (by 24% versus healthy control) of the fluorescent label in the lymphocytes of patients heterozygous for this mutation. Concentrations of serum LDL-C in probands before treatment were between 9.5 and 10.5 mmol/l. All patients had corneal arcus and tendon xanthoma. Clinically, families were characterized by premature coronary artery disease. This mutation occurred relatively frequently in our group of patients with FH, but this could be explained by a founder effect since we demonstrated their common ancestors.

Corticosteroid treatment of kidney disease in a patient with familial lecithin-cholesterol acyltransferase deficiency.

Familial lecithin-cholesterol acyltransferase (LCAT) deficiency (FLD) is a rare genetic disorder of lipid metabolism, characterised by low plasma HDL cholesterol, proteinuria, haemolytic anaemia and corneal opacities. Usually renal disease progresses during the third decade of life to renal failure; however the pathogenesis of renal disease is not well understood. In this study we describe treatment of renal disease in two siblings with FLD. The proband WX at the age of 31 years presented proteinuria and ankle oedema during her third pregnancy. Diagnosis of FLD was based on a renal biopsy with characteristic serpiginous fibrillar deposits under electron microscopy, markedly decreased HDL cholesterol, esterified cholesterol levels and LCAT activity, confirmed by molecular analysis. After 3 years her proteinuria increased and she received an ACE inhibitor to which she responded well. During further increases of proteinuria she additionally received methylprednisolone and her proteinuria decreased. This long-term observation indicates the efficacy of corticosteroids and renin-angiotensin-aldosterone system blockers in the treatment of proteinuria in patients with FLD. The results suggest the role of inflammatory processes as well as dyslipidemia in the pathogenesis of glomerular disorders in LCAT-deficient patients.