The molecular genetic background of familial hypercholesterolemia: data from the Slovak nation-wide survey.

Familial hypercholesterolemia (FH) is most frequently caused by LDLR or APOB mutations. Therefore, the aim of our study was to examine the genetic background of Slovak patients suspected of FH. Patients with clinical suspicion of FH (235 unrelated probands and 124 family relatives) were recruited throughout Slovakia during the years 2011-2015. The order of DNA analyses in probands was as follows: 1. APOB mutation p.Arg3527Gln by real-time PCR method, 2. direct sequencing of the LDLR gene 3. MLPA analysis of the LDLR gene. We have identified 14 probands and 2 relatives with an APOB mutation p.Arg3527Gln, and 89 probands and 75 relatives with 54 different LDLR mutations. Nine of LDLR mutations were novel (i.e. p.Asp90Glu, c.314-2A>G, p.Asp136Tyr, p.Ser177Pro, p.Lys225_Glu228delinsCysLys, p.Gly478Glu, p.Gly675Trpfs*42, p.Leu680Pro, p.Thr832Argfs*3). This is the first study on molecular genetics of FH in Slovakia encompassing the analysis of whole LDLR gene. Genetic etiology of FH was confirmed in 103 probands (43.8 %). Out of them, 86.4 % of probands carried the LDLR gene mutation and remaining 13.6 % probands carried the p.Arg3527Gln APOB mutation.

Molecular-genetic aspects of familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is the world's most abundant and the most common heritable disorder of lipid metabolism. The prevalence of the disease in general population is 1:500. Therefore the approximate number of FH patients all over the world is 14 million. From the genetic point of view the disease originates as a result of mutations in genes affecting the processing of LDL particles from circulation, resulting in an increase in LDL cholesterol and hence total cholesterol. These are mutations in genes encoding LDL receptor, apolipoprotein B, proprotein convertase subtilisin/kexin 9 and LDL receptor adaptor protein 1. Cholesterol depositing in tissues and blood vessels of individuals creates tendon xanthoma, xanthelesma and arcus lipoides cornae. Due to the increased deposition of cholesterol in blood vessels, atherosclerosis process is accelerated, what leads to a significantly higher risk of premature cardiovascular diseases. Therefore, early clinical diagnosis confirmed by the DNA analysis, and effective treatment are crucial to reduce the mortality and high risk of premature atherosclerotic complications.

Heritability, genetic correlation and linkage to the 9p21.3 region of mixed platelet-leukocyte conjugates in families with and without early myocardial infarction.

BACKGROUND AND AIMS: Variations in mixed platelet-leukocyte conjugate formation in human whole blood could be genetically determined. We quantified platelet and leukocyte activation and interaction in families with or without early myocardial infarction and evaluated their heritability, genetic correlation and linkage to the 9p21.3 region. METHODS AND RESULTS: The study population included 739 subjects (≥ 15 years old) from 54 large pedigrees, 23 with and 31 without familial myocardial infarction. Mixed platelet-leukocyte conjugates and markers of platelet or leukocyte activation (P-selectin, CD11b and L-selectin surface expression) were measured both before and after in vitro blood stimulation with collagen-ADP. All traits had significant genetic components (17.5-65.3% of the phenotypic variability), while shared household effects (0-39.6%) and environmental covariates (0-10.2%) tended to be smaller. Stimulated platelet-polymorphonuclear leukocyte (PMN) and platelet-monocyte conjugates showed the highest linkage to the 9p21.3 region (LOD = 0.94 and 1.33, respectively; empirical p value = 0.017 and 0.009). PMN markers resulted strongly genetically correlated between them in bivariate analysis among pairs of quantitative traits. CONCLUSION: This study supports a genetic regulation of human mixed platelet-leukocyte conjugates.

Association of metabolic and genetic factors with cholesterol esterification rate in HDL plasma and atherogenic index of plasma in a 40 years old Slovak population.

We assessed association between novel biomarkers of cardiovascular disease and conventional factors in 40 years old subjects (208 men and 266 women) from the general population of Slovakia. FER(HDL) (cholesterol esterification rate in HDL plasma), AIP--Atherogenic Index of Plasma [Log(TG/HDL-C)] as markers of lipoprotein particle size, and CILP2, FTO and MLXIPL polymorphisms, were examined in relation to biomarkers and conventional risk factors. Univariate analyses confirmed correlation between AIP, FER(HDL) and the most of measured parameters. Relations between AIP and CILP2, FTO and MLXIPL were not significant. However, CILP2 was significantly related to FER(HDL) in both genders. In multivariate analysis BMI was the strongest correlate of AIP levels. In multivariate model variability of FER(HDL) was best explained by AIP (R(2) = 0.55) in both genders with still significant effect of CILP2 SNP in men. In a model where AIP was omitted, TG levels explained 43 % of the FER(HDL) variability in men, while in women HDL-C was the major determinant (42 %). In conclusions, FER(HDL) and AIP related to the known markers of cardiovascular risk provide means to express their subtle interactions by one number. Our novel finding of association between CILP2 polymorphism and FER(HDL) supports its role in lipid metabolism.

Genetic regulation of inflammation-mediated activation of haemostasis: family-based approaches in population studies.

Blood coagulation and inflammation play a key role in atherosclerosis and thrombosis. Candidate gene and genome wide association studies have identified potential specific genes that might have a causal role in these pathogenic processes. The analysis of quantitative traits is more powerful as they are closer to direct gene action than disease phenotypes. Thus linkage-based studies on extended families might be useful both to estimate the heritability and to map the genetic loci responsible for the regulation of the trait. Family-based studies may estimate high heritability for thrombosis and quantitative traits regarding both platelet aggregation and blood coagulation. Some specific loci relevant to thrombosis have been identified, with some of them showing a direct pleiotropic effect on the risk of thrombosis. Haemostasis factors can be activated by inflammatory stimuli. Fibrinogen level is genetically correlated with C-reactive protein levels with a link for both traits on chromosomes 12 and 21. Genes related to prostanoid biosynthesis, involved both in inflammation and thrombosis, show high heritability levels in both enzyme expression and prostanoid production. Considering that few large family-based linkage studies have as yet been performed on haemostasis and inflammation-related traits, additional studies are highly needed. We are performing a family-based linkage study on large pedigrees (750 subjects from 23 families with juvenile myocardial infarction and 31 control families), to identify genes responsible for quantitative traits involved in the pathway progressively going from inflammation to haemostasis, cell activation, thrombus formation and cardiovascular events.

Evaluation of alternative calculation methods for determining LDL cholesterol.

BACKGROUND: Due to limitations of the Friedewald formula, alternative methods for calculating low-density lipoprotein cholesterol (LDL-C) were suggested. We evaluated utility of these methods. METHODS: Ninety three subjects free of coronary heart disease were considered. LDL-C was measured by the homogeneous method, and calculated by the Friedewald formula LDL-C = TC-HDL-(TG/2.2) (LDL1) and alternative formulas LDL-C = 0.41 TC - 0.32 TG + 1.70 apoB - 0.27 (LDL2) and LDL-C = 0.94 TC - 0.94 HDL - 0.435 TG (LDL3). RESULTS: All three formulas underestimated the measured LDL-C, both in the whole group and in subgroups according to TG levels (TG < 1.7 and in a range of 1.7-4.5 mmol/l, p < 0.001 for all). We found significantly higher bias for all three formulas in subjects with 1.7 < or = TG < 4.5 mmol/l levels. The Friedewald formula showed the lowest assay bias in all the groups investigated. The mean absolute bias for LDL1 was 7.6%, 18.3% for LDL2 and 13.6% for LDL3, respectively. Linear regression analysis showed correlation of calculated LDL-C values with the direct method in the range of r = 0.82 - 0.90 (p < 0.0001 for all, except of LDL2 in 1.7 < or = TG < 4.5 mmol/l group where p = 0.0011). CONCLUSIONS: The Friedewald formula seems to be a better estimator of LDL-C in our study than the other two alternative formulas; however, it underestimated the LDL-C levels.

Atherogenic lipoprotein profile in families with and without history of early myocardial infarction.

In this study we compared several parameters characterizing differences in the lipoprotein profile between members of families with a positive or negative family history of coronary artery disease (CAD). In addition to regular parameters such as the body mass index (BMI), total plasma cholesterol (TC), low density (LDL-C) and high density (HDL-C) cholesterol and triglycerides (TG) we estimated the fractional esterification rate of cholesterol in apoB lipoprotein-depleted plasma (FER(HDL)) which reflects HDL and LDL particle size distribution. A prevalence of smaller particles for the atherogenic profile of plasma lipoproteins is typical. Log (TG/HDL-C) as a newly established atherogenic index of plasma (AIP) was calculated and correlated with other parameters. The cohort in the study consisted of 29 young (< 54 years old) male survivors of myocardial infarction (MI), their spouses and at least one offspring (MI group; n=116). The control group consisted of 29 apparently healthy men with no family history of premature CAD in three generations, their spouses and at least one offspring (control group; n=124). MI families had significantly higher BMI than the controls, with the exception of spouses. Plasma TC did not significantly differ between MI and the controls. MI spouses had significantly higher TG. Higher LDL-C had MI survivors only, while lower HDL-C had both MI survivors and their spouses compared to the controls. FER(HDL) was significantly higher in all the MI subgroups (probands 25.85+/-1.22, spouses 21.55+/-2.05, their daughters 16.93+/-1.18 and sons 19.05+/-1.33 %/h) compared to their respective controls (men 20.80+/-1.52, spouses 14.70+/-0.98, daughters 13.23+/-0.74, sons 15.7+/-0.76 %/h, p<0.01 to p<0.05). Log(TG/HDL-C) ranged from negative values in control subjects to positive values in MI probands. High correlation between FER(HDL) and Log (TG/HDL-C) (r=0.80, p<0.0001) confirmed close interactions among TG, HDL-C and cholesterol esterification rate. The finding of significantly higher values of FER(HDL) and Log (TG/HDL-C) indicate higher incidence of atherogenic lipoprotein phenotype in members of MI families. The possibility that, in addition to genetic factors, a shared environment likely contributes to the familial aggregation of CAD risk factors is supported by a significant correlation of the FER(HDL) values within spousal pairs (control pairs: r=0.51 p<0.01, MI pairs: r=0.41 p<0.05).

Risk factors for atherosclerosis in survivors of myocardial infarction and their spouses: comparison to controls without personal and family history of atherosclerosis.

To explore the hypothesis that an interplay between genetic and environmental factors contributes to the development of coronary atherosclerosis, we compared the prevalence of risk factors for atherosclerosis among survivors of myocardial infarction (MI) and their spouses and apparently healthy men and women (spousal pairs) with no personal and family history of atherosclerosis in three generations. There were no significant differences in life-style and dietary habits between the groups. The daily vegetable and/or fruit intake was generally low and did not differ between the groups. Thirty percent and 25% of men and women did not consume any vegetables or fruits, respectively. All differences found in the male MI survivors and control men were also found between the female groups: MI survivors and their spouses were significantly more obese and had higher systolic and diastolic blood pressure and more pathologic plasma lipid levels compared with control males and females, respectively. Compared with the control men and women, MI survivors and spouses had higher plasma homocysteine (Hcgamma) levels (15.3 +/- 10.5, 11.9 +/- 4.0, 16.9 +/- 5.5, and 14.3 +/- 4.0, micromol/L, respectively, P = .01). The frequency of the homozygous C677T 5,10-methylenetetrahydrofolate reductase (MTHFR) polymorphism in MI survivors was twice that observed in their spouses and controls (12.1%, 4.8%, and 5.8%, respectively), but this difference did not reach statistical significance. A statistically significant association of the MTHFR genotype and Hcgamma concentration (multiple ANOVA) was shown. Neither the frequencies of apolipoprotein E (apoE) alleles nor Asp9Asn mutation of exon 2, Asn29lSer mutation of exon 6, and Ser447Ter of exon 9 of the lipoprotein lipase (LPL) gene varied significantly among the groups. A possible explanation for our findings is that individuals with a genetic predisposition for atherosclerosis and their spouses share a life-style that results in a higher body mass index (BMI) and waist to hip ratio (WHR). On the other hand, individuals with no family history of atherosclerosis, despite an unhealthy life-style similar to that in the affected families (diet and physical activity), had a lower BMI and WHR and more favorable metabolic parameters, including plasma Hcgamma. In conclusion, we have shown that a personal and/or family history of atherosclerosis corresponds to the prevalence and level of risk factors for atherosclerosis. A combination of life-style factors and inherited metabolic abnormalities, including high plasma Hcgamma, are the more likely explanation for our findings.

DNA damage in diabetes: correlation with a clinical marker.

Levels of DNA damage in groups of 10 patients with insulin-dependent diabetes mellitus and 10 matched controls were compared using the comet assay; DNA strand breaks, oxidized pyrimidines (endonuclease III-sensitive sites) and altered purines (sites sensitive to formamidopyrimidine glycosylase) were measured. Mean values of strand breaks and oxidized pyrimidines were significantly higher in diabetics. Strand breaks correlated with body mass index in the diabetic group. A strong correlation was seen between formamidopyrimidine glycosylase-sensitive sites and serum glucose concentrations. When three patients with normal glucose levels were excluded from the statistical analysis, the mean value of formamidopyrimidine glycosylase-sensitive sites was very significantly elevated compared with normal. DNA damage in lymphocytes is thus a useful marker of oxidative stress, and in particular formamidopyrimidine glycosylase-sensitive sites seem to represent changes specifically related to hyperglycemia.

Apolipoprotein E genotypes in offspring with a positive and negative family history of premature myocardial infarction.

Apolipoprotein E (apo-E) allele and genotype frequencies were evaluated in offspring with positive (MI-offspring) and negative (control-offspring) parental history of myocardial infarction (MI). The apo-E allele frequencies in MI- and control-offspring were as follows: epsilon2: 9.04 and 2.08% (p < 0.02), epsilon3: 84.04 and 87.5%, epsilon4: 6.91 and 10.41%, respectively. The frequencies of the E2-genotypes were significantly lower in offspring of controls (4.2%, 17.0%, respectively, p < 0.03). The epsilon2-allele is associated with raised plasma triglyceride concentrations in subjects on a diet high in saturated fat. We therefore hypothesize that offspring carrying an epsilon2-allele are predisposed to develop disturbance of plasma triglyceride metabolism when exposed to a traditional Slovak high-fat diet and/or weight gain, resulting in altered lipid levels and increased predisposition to atherosclerosis.