A number of chronic diseases, including cardiovascular disease, appear to have a multifactorial genetic risk component. Consequently, techniques are needed to facilitate evaluation of complex genetic risk factors in large cohorts. We have designed a prototype assay for genotyping a panel of 35 biallelic sites that represent variation within 15 genes from biochemical pathways implicated in the development and progression of cardiovascular disease. Each DNA sample is amplified using two multiplex polymerase chain reactions, and the alleles are genotyped simultaneously using an array of immobilized, sequence-specific oligonucleotide probes. This multilocus assay was applied to two types of cohorts. Population frequencies for the markers were estimated using 496 unrelated individuals from a family-based cohort, and the observed values were consistent with previous reports. Linkage disequilibrium between consecutive pairs of markers within the apoCIII, LPL, and ELAM genes was also estimated. A preliminary analysis of single and pairwise locus associations with severity of atherosclerosis was performed using a composite cohort of 142 individuals for whom quantitative angiography data were available; evaluation of the potentially interesting associations observed will require analysis of an independent and larger cohort. This assay format provides a research tool for studies of multilocus genetic risk factors in large cardiovascular disease cohorts, and for the subsequent development of diagnostic tests.

Several polymorphisms in the apolipoprotein C-III (apoC-III) gene have been associated with hypertriglyceridemia, but the link with coronary artery disease risk is still controversial. In particular, apoC-III promoter sequence variants in the insulin responsive element (IRE), constitutively resistant to downregulation by insulin, have never been investigated in this connection. We studied a total of 800 patients, 549 of whom had angiographically documented coronary atherosclerosis, whereas 251 had normal coronary arteriograms. We measured plasma lipids, insulin, apoA-I, apoB, and apoC-III and assessed three polymorphisms in the apoC-III gene, namely, T-455C in the IRE promoter region, C1100T in exon 3, and Sst1 polymorphic site (S1/S2) in the 3 untranslated region. Each variant influenced triglyceride levels, but only the T-455C (in homozygosity) and S2 alleles influenced apoC-III levels. In coronary artery disease (CAD) patients, 18.6% were homozygous for the ؊ 455C variant compared with only 9.2% in CAD-free group ( P Ͻ 0.001). In logistic regression models, homozygosity for ؊ 455C variant was associated with a significantly increased risk of CAD (OR ‫؍‬ 2.5 and 2.18 for unadjusted and adjusted models, respectively) suggesting that it represents an independent genetic susceptibility factor for CAD. -

Background and Purpose-The etiology of stroke in children with sickle cell anemia (SCA) is complex and poorly understood. Growing evidence suggests that genetic factors beyond the sickle cell mutation influence stroke risk in SCA. We previously reported risk associations with polymorphisms in several proinflammatory genes in SCA children with ischemic stroke. The aim of this replication study was to confirm our previous findings of associations between the TNF(Ϫ308) G/A, IL4R 503 S/P, and ADRB2 27 Q/E polymorphisms and large vessel stroke risk. Methods-Using previously collected MRA data, we assessed an independent population of SCA children from the multicenter Stroke Prevention Trial in Sickle Cell Anemia (STOP) for the presence or absence of large vessel stenosis. Samples were genotyped for 104 polymorphisms among 65 candidate vascular disease genes. Genotypic associations with risk of large vessel stroke were screened using univariable analysis and compared with results from our original study. Joint analysis of the 2 study populations combined was performed using multivariable logistic regression. Results-A total of 96 children (49 MRA-positive, 47 MRA-negative) were included in this study. Of the SNP associations previously identified in the original study, the TNF(Ϫ308) G/A association with large vessel stroke remained significant and the IL4R 503 S/P variant approached significance in the joint analysis of the combined study populations. Consistent with our original findings, the TNF(Ϫ308) GG genotype was associated with a Ͼ3-fold increased risk of large vessel disease (ORϭ3.27; 95% CIϭ1.6, 6.9; Pϭ0.006). Unadjusted analyses also revealed a previously unidentified association between the LTC4S(Ϫ444) A/C variant and large vessel stroke risk. Conclusions-Similar findings in 2 independent study populations strongly suggest that the TNF(Ϫ308) G/A promoter polymorphism is a clinically important risk factor for large vessel stroke in children with SCA. The previously observed association with the IL4R 503 S/P variant and the novel association with the LTC4S(Ϫ444) A/C variant suggest that these loci may also contribute to large vessel stroke risk in children with SCA.

Several compelling lines of evidence suggest an important influence of genetic variation in susceptibility to Kawasaki disease (KD), an acute vasculitis that causes coronary artery aneurysms in children. We performed a family-based genotyping study to test for association between KD and 58 genes involved in cardiovascular disease and inflammation. By analysis of a cohort of 209 KD trios using the transmission disequilibrium test, we documented the asymmetric transmission of five alleles including the interleukin-4 (IL-4) C(-589)T allele (P=0.03). Asymmetric transmission of the IL-4 C(-589)T was replicated in a second, independent cohort of 60 trios (P=0.05, combined P=0.002). Haplotypes of alleles in IL-4, colony-stimulating factor 2 (CSF2), IL-13, and transcription factor 7 (TCF7), all located in the interleukin gene cluster on 5q31, were also asymmetrically transmitted. The reported associations of KD with atopic dermatitis and allergy, elevated serum IgE levels, eosinophilia, and increased circulating numbers of monocyte/macrophages expressing the low-affinity IgE receptor (FCepsilonR2) may be related to effects of IL-4. Thus, the largest family-based genotyping study of KD patients to date suggests that genetic variation in the IL-4 gene, or regions linked to IL-4, plays an important role in KD pathogenesis and disease susceptibility.

The complexity of recognizing the potential contribution of a number of possible predictors of complex disorders is increasingly challenging with the application of large-scale single nucleotide polymorphism (SNP) typing. In the search for putative genetic factors predisposing to coronary artery restenosis following balloon angioplasty, we determined genotypes for 94 SNPs representing 62 candidate genes, in a prospectively assembled cohort of 342 cases and 437 controls. Using a customized coupled-logistic regression procedure accounting for both additive and interactive effects, we identified seven SNPs in seven genes that, together, showed a statistically significant association with restenosis incidence (P Ͻ0.0001), accounting for 11.6% of overall variance observed. Among them are candidate genes for cardiovascular pathophysiology (apolipoprotein-species and NOS), inflammatory response (TNF receptor and CD14), and cell-cycle control (p53 and p53-associated protein). Our results emphasize the need to account for complex multi-gene influences and interactions when assessing the molecular pathology of multifactorial medical entities.

In our efforts to develop diagnostic tests for complex multifactorial disorders, and to assist the research community in evaluating genetic markers for predisposition to cardiovascular disease, we have developed a prototype assay to genotype up to 35 variable sites among 15 genes. The candidate markers in this panel were selected from biological pathways likely to contribute to the development and progression of cardiovascular disease. Each sample is amplified in two multiplex polymerase chain reactions that are then hybridized to an array of immobilized oligonucleotide probes. The assay has been applied to a population-based cohort representing 238 families; allele frequencies observed among 455 unrelated parents from this cohort agree with available literature values. Data from a cohort of 142 lipid-clinic patients were used to explore locus associations with arterial occlusion, as measured by quantitative angiography. This prototype assay provides a research tool for studies to assess the association of multiple markers with disease, and for clinical studies to evaluate marker association with patient responsiveness to experimental therapies.

Type 1 diabetes is associated with coronary heart disease (CHD) and coronary artery calcification (CAC), a measure of subclinical CHD. The hepatic lipase gene promoter polymorphism (LIPC-480C>T) is a common variant affecting lipid metabolism. This study examined the relation between the LIPC-480C>T and CAC in type 1 diabetes. In the type 1 diabetic patients studied, 56% had CAC >0 Agatston units (AU). These subjects had a longer duration of diabetes (26.2 ؎ 1.3 vs. 17.8 ؎ 1.4 years; P < 0.001), lower HDL cholesterol levels (55.7 ؎ 2.4 vs. 61.0 ؎ 2.5 mg/dl; P ‫؍‬ 0.05), higher triglyceride levels (101 ؎ 17.3 vs. 66 ؎ 7.6 mg/dl; P < 0.05), and higher diastolic blood pressure (79.7 ؎ 1.0 vs. 76.0 ؎ 1.4 mmHg; P < 0.05). The LIPC-480 T allele was more common in subjects with CAC (frequency ‫؍‬ 0.31 ؎ 0.05 vs. 0.14 ؎ 0.04; P ‫؍‬ 0.006). The proportion with CAC was 44% in LIPC-480CC subjects, 71% in heterozygotes, and 83% in LIPC-480TT subjects (P < 0.01). LIPC-480 T allele frequency increased as the amount of CAC increased (P ‫؍‬ 0.007). LIPC-480 genotype was independently associated with the CAC (odds ratio ‫؍‬ 2.90, 95% CI 1.22-6.92, P < 0.05) after adjusting for duration of diabetes, age, sex, diastolic blood pressure, HDL cholesterol, and triglyceride levels. In conclusion, the LIPC-480C>T polymorphism was associated with subclinical CHD in type 1 diabetes. This genetic variant may identify subjects in which early intervention to prevent CHD may be appropriate.

Variants in mannose-binding lectin (MBL2; protein MBL) have shown association with different aspects (eg, lung function, infection, survival) of cystic fibrosis (CF) in some studies but not others. Inconsistent results may be due to confounding among disease variables that were not fully accounted for in each study. To account for these relationships, we derived a modeling framework incorporating CFTR genotype, age, Pseudomonas aeruginosa (Pa) infection, and lung function from 788 patients in the US CF Twin and Sibling Study. This framework was then used to identify confounding variables when testing the effect of MBL2 variation on specific CF traits. MBL2 genotypes corresponding to low levels of MBL associated with Pa infection 1.94 years earlier than did MBL2 genotypes corresponding to high levels of MBL (P=0.0034). In addition, Pa-infected patients with MBL2 genotypes corresponding to low levels of MBL underwent conversion to mucoid Pa 2.72 years earlier than did patients with genotypes corresponding to high levels of MBL (P=0.0003). MBL2 was not associated with the time to transition from infection to conversion or with lung function. Thus, use of a modeling framework that identified confounding among disease variables revealed that variation in MBL2 associates with age at infection with Pa and age at conversion to mucoid Pa in CF.