Testing for lipoprotein(a) concentration and apolipoprotein(a) phenotypes: method standardization and pediatric reference values.

Increased levels of lipoprotein(a) (Lp[a])are known independent risk factor for atherosclerosis, heart disease, and stroke in adults. Even in children it could be shown that elevated levels of Lp(a) are an independent risk factor for symptomatic thromboembolism. The aim of this work was to describe the methods used for evaluating Lp(a) phenotypes, to link them to Lp(a) plasma concentrations, and to establish age-dependent reference values in children. Lp(a) plasma concentrations were measured with enzyme-linked immunosorbent assay technique in parallel to agarose gel electrophoresis and subsequent anti-apolipoprotein(a) immunoblotting. We included 184 pediatric patients with stroke or venous thromboembolism, and 150 healthy age-matched controls in this study. In the control children we could find a mean Lp(a) concentration of 3 mg/dL for children 1 to 12 months of age, and in subjects 1.2 to 18 years of age, the mean Lp(a) concentration was 10 mg/dL. Using percentile classification the upper percentile cut-offs were as follows: age 3 to 6 months: 14 mg/dL; 6.1 to 12 months: 15 mg/dL; 1.1 to 9 years: 22 mg/dL; and 9.1 to 18 years: 30 mg/dL, respectively. In the present study we have established age-dependent reference values of plasma Lp(a) concentrations. The latter will help to harmonize international pediatric studies and to further evaluate the role of elevated Lp(a) in childhood vascular disease.

Fibrinogen alpha and gamma genes and factor VLeiden in children with thromboembolism: results from 2 family-based association studies.

Previous case-control studies showed that genetic variation in the fibrinogen gamma gene (FGG) increased the risk for deep vein thrombosis (VT) in adults. We investigated the association between the fibrinogen alpha (FGA) and FGG haplotypes, the factor V(Leiden)-mutation, and pediatric VT and thromboembolic stroke (TS) in 2 independent study samples. Association analysis revealed that the FGA-H1 and FGG-H2 haplotypes were significantly overtransmitted to VT patients (FGA-H1, P = .05; FGG: H2, P = .032). In contrast, the FGG-H3 haplotype was undertransmitted (P = .022). In an independent study sample, FGA-H1 (P = .008) and FGG-H2 (P = .05) were significantly associated with TS. The association of FGA and FGG haplotypes with VT was more pronounced in FV(Leiden)-negative families (FGA-H1, P = .001; FGG-H2, P = .001), indicating a genetic interaction between both risk factors. The risk-conferring FGG-H2 and the protective FGG-H3 haplotypes correlated with low (FGG-H2) and high (FGG-H3) levels of the gamma' chain variant, respectively. These results provide independent and novel evidence that FGA-H1 and FGG-H2 variants are associated with an increased risk of VT and TS in children. The observed negative correlation of genetic VT risk with the plasma levels of the fibrinogen gamma' variant suggests that FGG-H2 and -H3 haplotypes modify thrombosis risk by controlling the level of this FGG splice isoform.

Association of the protein Z ATG haplotype with symptomatic nonvascular stroke or thromboembolism in white children: a family-based cohort study.

To clarify the role of protein Z (PZ) in children with stroke/thromboembolism (TE), the present haplotype (HT)-based family study was performed. We genotyped 365 pediatric stroke/TE families (stroke n = 216; TE n = 149) for 4 single nucleotide polymorphisms (SNPs; rs3024718, rs3024731, rs3024772, and rs3024778) to assess the association between genetic variation within a conserved block of linkage disequilibrium harboring the PZ gene and pediatric TE. Association was assessed with use of the transmission disequilibrium test (TDT), corrected for multiple testing (permutation testing: HAPLOVIEW). In addition, PZ antigen was determined and correlated with carriership of PZ haplotypes and the FV G1691A mutation. Rs3024718, rs3024731, and rs3024772 are in tight linkage disequilibrium (LD) and define 4 haplotypes, capturing 97% of the genetic variation for this LD block. HT1 (ATG) was significantly overtransmitted from parents to affected offspring (HT frequency 73.5%, T:U 122:80, chi(2) = 8.791, P = .003). The ATG risk haplotype was significantly correlated with greater PZ antigen levels. Multivariate analysis adjusted for age, sex, established thrombophilias, smoking, fibrinogen, and PZ levels revealed a significant association of the ATG haplotype and TE in children (odds ratio [OR] 1.4; 95% confidence interval [95% CI] 1.08-1.93). Our results suggest that the ATG haplotype of the PZ gene is a genetic marker for symptomatic TE in white German children.

Genetics of hemostasis: differential effects of heritability and household components influencing lipid concentrations and clotting factor levels in 282 pediatric stroke families.

BACKGROUND: The identification of heritable and environmental factors possibly influencing a condition at risk should be a prerequisite for the search for the proportion of variance attributable for shared environmental effects (c(2)) modulating the risk of disease. Such epidemiologic approaches in families with a first acute ischemic stroke during early childhood are lacking. OBJECTIVES: Our goal was to estimate the phenotypic variation within lipid concentrations and coagulation factor levels and to estimate the proportions attributable to heritability (h(2)r) and c(2) in pediatric stroke families. METHODS: Blood samples were collected from 1,002 individuals from 282 white stroke pedigrees. We estimated h(2)r and c(2) for lipoprotein (a) [Lp(a)], cholesterol, high-density lipoprotein, low-density lipoprotein (LDL), fibrinogen, factor (F) II, FV, FVIIIC, von Willebrand factor (vWF), antithrombin, protein C, protein S, plasminogen, protein Z, total tissue factor pathway inhibitor (TFPI), prothrombin fragment F1.2, and D-dimer, using the variance component method in sequential oligogenetic linkage analysis routines. RESULTS: When incorporating h(2)r and c(2) in one model adjusted for age, blood group, sex, smoking, and hormonal contraceptives, significant h(2)r estimates were found for Lp(a), LDL, fibrinogen, protein C, and protein Z. In addition to the significant h(2)r estimates, c(2) showed a significant effect on phenotypic variation for fibrinogen, protein C, and protein Z. A significant c(2) effect was found for cholesterol, and plasma levels of FII, FV, vWF, antithrombin, protein S, plasminogen, and TFPI, ranging from 9.3% to 33.2%. CONCLUSIONS: Our research stresses the importance of research on the genetic variability and lifestyle modifications of risk factors associated with pediatric stroke.

Elevated alpha1-antitrypsin is a risk factor for arterial ischemic stroke in childhood.

Alpha1-antitrypsin (alpha1-AT) is a physiological inhibitor of activated protein C (APC) and therefore decreased APC activity. APC itself causes an anticoagulant effect by inactivating factors Va and VIIIa. The present case-control study was performed to evaluate the role of the elevated alpha1-AT concentration in pediatric patients with ischemic stroke (IS). alpha1-AT concentrations were measured along with established prothrombotic risk factors 6-12 months after the acute thrombotic onset in 81 Caucasian children with IS aged 1 month to 18 years. The cutoff values defined as age-dependent 90th percentiles were obtained from 229 healthy controls. Median (range) values of alpha1-AT were significantly higher in patients compared with control subjects [122.0 mg/dl (61.4-224.0) vs. 114.0 mg/dl (66.8-156.0); p = 0.016]. In addition, 14 of the 81 patients (17.3%) compared with 10 of the 162 controls (6.2%) had alpha1-AT concentrations above the 90th age-dependent percentiles (p = 0.012). Multivariate analysis performed in a 1:2 matched case-control setting adjusted for the presence of established prothrombotic risk factors showed a significantly increased odds ratio (OR) and 95% confidence interval (CI) for patients with elevated alpha1-AT >90th percentiles and IS (OR/CI: 4.0/1.64-9.92; p = 0.0024). Data shown here give evidence that total alpha1-AT concentrations above the 90th age-dependent percentiles independently increase the risk of IS 4.0-fold in Caucasian children.

Total tissue factor pathway inhibitor is an independent risk factor for symptomatic paediatric venous thromboembolism and stroke.

Tissue factor pathway inhibitor (TFPI) plays an important role in inhibiting tissue factor-induced coagulation by a factor Xadependent pathway of the activated tissue-factor VIIa complex. Decreased values of the latter inhibitor have been recently reported in adult patients with venous thrombosis (VT) or ischaemic stroke (IS). The present case-control study was therefore performed to evaluate whether a decreased TFPI concentration is also involved in paediatric symptomatic thromboembolism (ST). Total TFPI concentrations were measured along with established prothrombotic risk factors six to twelve months after the acute thrombotic onset in 144 Caucasian children aged 0.6 to 18 years (VT: n=80; IS: n=64). The cut-off values defined as age-dependent 10(th) percentiles were obtained from 244 healthy controls. Median (range) values of TFPI were significantly lower in patients compared with control subjects [50.0(20.0-132.3) ng/ml vs. 59.5(25.4-117.4) ng/ml; p-value < 0.0001]. In addition, 42 of the 144 patients (29.2%) compared with 25 of the 244 controls (10.2%) showed TFPI concentrations below the 10(th) age-dependent percentiles. Compared to baseline values 78.6% of children with total TFPI Ag < 10(th) percentiles showed a low response to enoxaparin administration, whereas in children with normal baseline TFPI values 30% show a low TFPI release (p = 0.007). Multivariate analysis adjusted for the presence of established prothrombotic risk factors showed a significantly increased odds ratio (OR) and 95% confidence interval (CI) for patients with ST [OR/CI: 3.8/2.2-6.6; p < 0.0001]. Data shown here give evidence that total TFPI concentrations below the 10(th) age-dependent percentiles independently increase the risk of ST in Caucasian children 3.8-fold.