Identification of a novel locus for triglyceride on chromosome 1p31-32 in families with premature CAD and MI

An increased plasma triglyceride (TG) level is associated with coronary artery disease (CAD) and myocardial infarction (MI) and is a key characteristic of the metabolic syndrome. Here, we used a genome-wide linkage scan to identify a novel genetic locus that influences the plasma TG level. We genotyped 714 persons in 388 multiplex Caucasian families with premature CAD and MI with 408 polymorphic microsatellite markers that cover the entire human genome. The genome-wide scan identified positive linkage for the quantitative TG trait to a novel locus on chromosome 1p31-32 [peak single-point logarithm of odds (LOD) = 3.57, peak multipoint LOD = 3.12]. For single-point linkage analysis, two markers, D1S1728 and D1S551, showed LOD scores of 2.42 and 3.57, respectively. For multipoint linkage analysis, three markers, D1S3736, D1S1728, and D1S551, showed LOD scores of 2.43, 3.03, and 3.12, respectively. No other chromosomal regions showed a LOD score of >2.2. This study identifies a new genetic locus for TG on chromosome 1p31-32. Future studies of the candidate genes at this locus will identify a specific gene influencing the TG, which will provide insights into novel regulatory mechanisms of TG metabolism and may be important for the development of therapies to prevent CAD.     

Genetic determinants of phenotypic diversity in humans

New technologies for rapidly assaying DNA sequences have revealed that the degree and nature of human genetic variation is far more complex then previously realized. These same technologies have also resulted in the identification of common genetic variants associated with more than 30 human diseases and traits.     

Wnt5a and Wnt5b exhibit distinct activities in coordinating chondrocyte proliferation and differentiation

Proper longitudinal growth of long bones relies on the regulation of specific spatial patterns of chondrocyte proliferation and differentiation. We have studied the roles of two members of the Wnt family, Wnt5a and Wnt5b in long bone development. We show that Wnt5a is required for longitudinal skeletal outgrowth and that both Wnt5a and Wnt5b regulate the transition between different chondrocyte zones independently of the Indian hedgehog (Ihh)/parathyroid hormone-related peptide (PTHrP) negative feedback loop. We find that important cell cycle regulators such as cyclin D1 and p130, a member of the retinoblastoma family, exhibit complimentary expression patterns that correlate with the distinct proliferation and differentiation states of chondrocyte zones. Furthermore, we show that Wnt5a and Wnt5b appear to coordinate chondrocyte proliferation and differentiation by differentially regulating cyclin D1 and p130 expression, as well as chondrocyte-specific Col2a1 expression. Our data indicate that Wnt5a and Wnt5b control the pace of transitions between different chondrocyte zones.     

An LRP8 variant is associated with familial and premature coronary artery disease and myocardial infarction

Our previous genomewide linkage scan of 428 nuclear families (GeneQuest) identified a significant genetic susceptibility locus for premature myocardial infarction (MI) on chromosome 1p34-36. We analyzed candidate genes in the locus with a population-based association study involving probands with premature coronary artery disease (CAD) and/or MI from the GeneQuest families (381 cases) and 560 controls without stenosis detectable by coronary angiography. A nonconservative substitution, R952Q, in LRP8 was significantly associated with susceptibility to premature CAD and/or MI by use of both population-based and family-based designs. Three additional white populations were used for follow-up replication studies: another independent cohort of CAD- and/or MI-affected families (GeneQuest II: 441 individuals from 22 pedigrees), an Italian cohort with familial MI (248 cases) and 308 Italian controls, and a separate Cleveland GeneBank cohort with sporadic MI (1,231 cases) and 560 controls. The association was significantly replicated in two independent populations with a family history of CAD and/or MI, the GeneQuest II family-based replication cohort and the Italian cohort, but not in the population with sporadic disease. The R952Q variant of LRP8 increased activation of p38 mitogen-activated protein kinase by oxidized low-density lipoprotein. This extensive study, involving multiple independent populations, provides the first evidence that genetic variants in LRP8 may contribute to the development of premature and familial CAD and MI.     

Mechanisms of guanosine triphosphate hydrolysis by Ras and Ras-GAP proteins as rationalized by ab initio QM/MM simulations

The hydrolysis reaction of guanosine triphosphate (GTP) by p21(ras) (Ras) has been modeled by using the ab initio type quantum mechanical-molecular mechanical simulations. Initial geometry configurations have been prompted by atomic coordinates of the crystal structure (PDBID: 1QRA) corresponding to the prehydrolysis state of Ras in complex with GTP. Multiple searches of minimum energy geometry configurations consistent with the hydrogen bond networks have been performed, resulting in a series of stationary points on the potential energy surface for reaction intermediates and transition states. It is shown that the minimum energy reaction path is consistent with an assumption of a two-step mechanism of GTP hydrolysis. At the first stage, a unified action of the nearest residues of Ras and the nearest water molecules results in a substantial spatial separation of the gamma-phosphate group of GTP from the rest of the molecule (GDP). This phase of hydrolysis process proceeds through the low barrier (16.7 kcal/mol) transition state TS1. At the second stage, the inorganic phosphate is formed in consequence of proton transfers mediated by two water molecules and assisted by the Gln61 residue from Ras. The highest transition state at this segment, TS3, is estimated to have an energy 7.5 kcal/mol above the enzyme-substrate complex. The results of simulations are compared to the previous findings for the GTP hydrolysis in the Ras-GAP (p21(ras)-p120(GAP)) protein complex. Conclusions of the modeling lead to a better understanding of the anticatalytic effect of cancer causing mutation of Gln61 from Ras, which has been debated in recent years.     

Systemic elevations of free radical oxidation products of arachidonic acid are associated with angiographic evidence of coronary artery disease

Oxidant stress is widely believed to participate in cardiovascular disease pathogenesis. However, progress in defining appropriate systemic antioxidant targeted therapies has been hindered by uncertainty in defining clinically relevant systemic oxidant stress measures. In a case control study, 50 subjects with CAD (>50% stenosis in one or more major coronary vessels) and 54 without CAD (<30% stenosis in all major coronary vessels) were tested. Plasma was isolated and stored under conditions designed to prevent artificial lipid peroxidation. Systemic levels of multiple (n=9) specific fatty acid oxidation products including individual hydroxyoctadecadienoic acids (HODEs), hydroxyeicosatetraenoic acids (HETEs), and F(2)-isoprostanes were simultaneously measured by high-performance liquid chromatography (HPLC) with on-line tandem mass spectrometry, along with traditional risk factors and C-reactive protein (CRP) levels. Of the markers monitored, only 9-HETE and F(2)-isoprostanes, both products of free radical-mediated arachidonic acid oxidation, were significantly elevated in patients with angiographically defined CAD (9-HETE, 8.7 +/- 4 vs 6.8 +/- 4 micromol/mol arachidonate, P = 0.011; and F(2)-isoprostanes, 9.4 +/- 5 vs 6.2 +/- 3 micromol/mol arachidonate, P < 0.001). In multivariable analyses with simultaneous adjustment for Framingham risk score and C-reactive protein, 9-HETE (4th quartile OR = 4.8, 95% CI=1.3 to 17.1; P = 0.016) and F(2)-isoprostanes (4th quartile OR=9.7, 95% CI=2.56 to 36.9; P < 0.001) remained strong and independent predictors of CAD risk. Systemic levels of 9-HETE and F(2)-isoprostanes are independently associated with angiographic evidence of CAD and appear superior to other specific oxidation products of arachidonic and linoleic acids as predictors of the presence of angiographically evident coronary artery disease.     

Mechanism of the chemical step for the guanosine triphosphate (GTP) hydrolysis catalyzed by elongation factor Tu

Elongation factor Tu (EF-Tu), the protein responsible for delivering aminoacyl-tRNAs (aa-tRNAs) to ribosomal A site during translation, belongs to the group of guanosine-nucleotide (GTP/GDP) binding proteins. Its active 'on'-state corresponds to the GTP-bound form, while the inactive 'off'-state corresponds to the GDP-bound form. In this work we focus on the chemical step, GTP+H(2)O-->GDP+Pi, of the hydrolysis mechanism. We apply molecular modeling tools including molecular dynamics simulations and the combined quantum mechanical-molecular mechanical calculations for estimates of reaction energy profiles for two possible arrangements of switch II regions of EF-Tu. In the first case we presumably mimic binding of the ternary complex EF-Tu.GTP.aa-tRNA to the ribosome and allow the histidine (His85) side chain of the protein to approach the reaction active site. In the second case, corresponding to the GTP hydrolysis by EF-Tu alone, the side chain of His85 stays away from the active site, and the chemical reaction GTP+H(2)O-->GDP+Pi proceeds without participation of the histidine but through water molecules. In agreement with the experimental observations which distinguish rate constants for the fast chemical reaction in EF-Tu.GTP.aa-tRNA.ribosome and the slow spontaneous GTP hydrolysis in EF-Tu, we show that the activation energy barrier for the first scenario is considerably lower compared to that of the second case.     

Multi-allelic haplotype association identifies novel information different from single-SNP analysis: A new protective haplotype in the LRP8 gene is against familial and early-onset CAD and MI

Our previous studies identified a functional SNP, R952Q in the LRP8 gene, that was associated with increased platelet activation and familial and early-onset coronary artery disease (CAD) and myocardial infarction (MI) in American and Italian Caucasian populations. In this study, we analyzed four additional SNPs near R952Q (rs7546246, rs2297660, rs3737983, rs5177) to identify a specific LRP8 SNP haplotype that is associated with familial and early-onset CAD and MI. We employed a case–control association design involving 381 premature CAD and MI probands and 560 controls in GeneQuest, 441 individuals from 22 large pedigrees in GeneQuest II, and 248 MI patients with family history and 308 controls in an Italian cohort. Like R952Q, LRP8 SNPs rs7546246, rs2297660, rs3737983, and rs5177 were significantly associated with early-onset CAD/MI in both population-based and family-based association studies in GeneQuest. The results were replicated in the GeneQuest II family-based population and the Italian population. We then carried out a haplotype analysis for all five SNPs including R952Q. One common haplotype (TCCGC) was significantly associated with CAD (P = 4.0 × 10^− 11) and MI (P = 6.5 × 10^− 12) in GeneQuest with odds ratios of 0.53 and 0.42, respectively. The results were replicated in the Italian cohort (P = 0.004, OR = 0.71). The sib-TDT analysis also showed significant association between the TCCGC haplotype and CAD in GeneQuest II (P = 0.001). These results suggest that a common LRP8 haplotype TCCGC confers a significant protective effect on the development of familial, early-onset CAD and/or MI.