Interaction between metabolic syndrome and PON1 polymorphisms as a determinant of the risk of coronary artery disease.

The enzyme serum paraoxonase plays an important role in antioxidant defences and prevention of atherosclerosis. Metabolic syndrome (MS) is a clinical condition associated with increased oxidant stress and cardiovascular mortality. Two common polymorphisms of serum paraoxonase, PON1 Leu(55)Met and Gln(192)Arg, have been postulated to modulate the cardiovascular risk. We studied 915 subjects with angiographic documentation: 642 subjects with coronary atherosclerosis and 273 with normal coronary arteries. Two hundred and twenty-four subjects met the diagnostic criteria of MS. We found a significant interaction between MS and both the PON1 polymorphisms in determining the risk of coronary artery disease (P<0.05 by likelihood-ratio test). The 55Leu and the 192Arg alleles, associated with reduced protection against lipid peroxidation, were associated with coronary artery disease only in the MS subgroup. Subjects with MS and both 55Leu and 192Arg alleles had significantly increased risk (OR=9.38 with 95% CI=3.02-29.13 after adjustment by multiple logistic regression) as compared to subjects without MS and with 55Met/Met-192Gln/Gln genotype. No increased risk was found for subjects with MS and the 55Met/Met-192Gln/Gln genotype. This study highlights a potential example of genetic (paraoxonase polymorphisms)-clinical (MS) interaction influencing cardiovascular risk.

Family-based association analysis implicates IL-4 in susceptibility to Kawasaki disease.

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.

Interaction between smoking and PON2 Ser311Cys polymorphism as a determinant of the risk of myocardial infarction.

BACKGROUND: Increased oxidative stress is thought to play a role in the pathogenesis of the atherothrombotic process. Paraoxonases (PONs) are closely related antioxidant enzymes encoded by clustered genes on chromosome 7q. We evaluated three PON polymorphisms (PON1 Leu55Met and Gln192Arg; PON2 Ser311Cys) as possible risk factors for coronary atherosclerotic disease (CAD) and/or its main thrombotic complication, myocardial infarction (MI). MATERIALS AND METHODS: We studied 890 subjects with angiographic documentation of coronary vessels (272=CAD-free; 618=CAD). In the CAD group, 341 subjects had a previous MI. RESULTS: Frequencies of various genotypes were not significantly different between CAD-free subjects and the entire CAD population. In the latter group, there were more carriers of the PON2 311Cys variation among those who had suffered a MI than among those who had not (P<0.01 by chi2). The adjusted OR for MI among PON2 311Cys carriers was 1.5 (95%CI, 1.03-2.19). A gene-environmental interaction was found between PON2 Ser311Cys and smoking. Smoking by itself was associated with an increased MI risk. Among smokers, however, the MI risk was related to PON2 genotype: Cys/Cys homozygotes (OR=5.3; 95%CI, 1.7-16.4) and Ser/Cys heterozygotes (OR=2.1; 95%CI, 1.3-3.6) were at greater risk than Ser/Ser subjects (OR=1.2; 95%CI, 0.8-1.8). The PON2 polymorphism did not influence the MI risk among nonsmokers. CONCLUSIONS: In CAD subjects, a proportion of the risk of MI may be influenced by the interaction between smoking and a polymorphism in the antioxidant enzyme PON2.

Multi-locus interactions predict risk for post-PTCA restenosis: an approach to the genetic analysis of common complex disease.

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 <0.0001), 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.

Genetic influences on lipid metabolism trait variability within the Stanislas Cohort.

The contribution of 17 polymorphisms within 13 candidate genes on lipid trait variability was investigated by a multiplex assay in 772 men and 780 women coming for a health checkup examination. The studied genes were APOE, APOB, APOC3, CETP, LPL, PON, MTHFR, FGB, GpIIIa, SELE, ACE, and AGT. We found that APOB-Thr71Ile, APOE-(112/158), APOC3-1100C/T, and SELE-98G/T polymorphisms had a significant effect on lipid traits (P < or = 0.001 to P < or = 0.01). Genetic effects accounted for 3.5-5.7% of variation in apolipoprotein B (apoB)-related traits among men, and for 5.7-9.0% among women. The contribution of APOE polymorphism on apoB-related traits variability was two to three times more important in women than in men. We found suggestive evidence for interactive effects between genetics and age, smoking status, and oral contraceptives. Increase of LDL-cholesterol and apoB concentrations with age was stronger among the epsilon4 carriers in women, and apolipoprotein A-I (apoA-I) concentration decreased with age in epsilon4 male carriers. The effect of epsilon2 allele on LDL-cholesterol was more important in the oral contraceptive users. In nonsmokers only, the APOC3-1100C allele in women was related to lower apoB-related traits concentrations, and in men to higher apoA-I and HDL-cholesterol concentrations. In conclusion, this work, in addition to the reinforcement of the already known associations between APOB, APOE, and APOC3 genes and lipids, leads to new perspectives in the complex relationships among genes and environmental factors. The newly observed relationships between E-selectine gene and lipid concentrations support the hypotheses of multiple metabolic pathways contributing to the complexity of lipids variability.

A novel multilocus genotyping assay to identify genetic predictors of stroke in sickle cell anaemia.

We describe a novel multilocus genotyping assay permitting simultaneous identification of 60 candidate markers for stroke in sickle cell anaemia (SCA). Based on cerebral magnetic resonance imaging (MRI), 69 patients were divided into stroke and control groups. The variant allele, CBS 278thr, showed protection from stroke, whereas the apoE3 allele showed a trend towards association with increased stroke risk. Several other variant alleles [TNFalpha (-308)A, CETP (-628)A, apoCIII (-641)A] showed a trend towards significant associations with stroke risk. These preliminary results on a small group of patients suggest that a multilocus genotyping assay may be valuable in identifying genes that increase the risk of stroke in SCA.

Familial studies on the genetics of cardiovascular diseases: the Stanislas cohort.

In a given individual, the level of cardiovascular risk results from the combination of and interactions between genetic and environmental components. We choose to investigate segregation analysis of intermediate phenotypes in healthy nuclear families, belonging to the Stanislas cohort, a large familial cohort composed of 1006 families, which will be followed for 10 years. We developed a panel of 35 genetic markers including genes involved in lipid metabolism, regulation of blood pressure, thrombosis, platelet function, and endothelial cell adhesion. The allele frequencies of the studied polymorphisms were in agreement with those reported in other Caucasian populations. As an example of segregation analysis, we investigated carotid intima-media thickness (CIMT) variability in a subset sample of the Stanislas cohort. We found that about 30% of CIMT variability was attributable to genetic factors. Associations between CIMT and polymorphisms in apo CIII, cholesteryl ester transfer protein, methylene tetrahydrofolate reductase, and fibrinogen genes were observed and explained about 20% of CIMT variability in men. Furthermore, as another example of association studies, we investigated the relations between E-selectin polymorphisms and blood pressure interindividual variability and longitudinal changes in unrelated adults of this familial population. The E-selectin Phe554 allele was found associated with lower systolic blood pressure and diastolic blood pressure.

Transient cardiac expression of the tinman-family homeobox gene, XNkx2-10.

In Drosophila, the tinman homeobox gene is absolutely required for heart development. In the vertebrates, a small family of tinman-related genes, the cardiac NK-2 genes, appear to play a similar role in the formation of the vertebrate heart. However, targeted gene ablation of one of these genes, Nkx2-5, results in defects in only the late stages of cardiac development suggesting the presence of a rescuing gene function early in development. Here, we report the characterization of a novel tinman-related gene, XNkx2-10, which is expressed during early heart development in Xenopus. Using in vitro assays, we show that XNkx2-10 is capable of transactivating expression from promoters previously shown to be activated by other tinman-related genes, including Nkx2-5. Furthermore, Xenopus Nkx2-10 can synergize with the GATA-4 and SRF transcription factors to activate reporter gene expression.

A multilocus genotyping assay for candidate markers of cardiovascular disease risk.

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.

Multilocus approach to cardiovascular risk.

Until now, our familial studies have showed that shared genetic and environmental factors are involved on lipid parameters variability. More precisely, being working on 119 families we have showed that: a) The apolipoprotein E (apo E) common polymorphism is involved in the total cholesterol, low density lipoprotein cholesterol (LDL-Chol), apo E, apo B levels variability, b) the apolipoprotein A-IV gene had no effect on lipid metabolism parameters variability, apo A-IV levels included, c) the apolipoprotein B gene was associated with total cholesterol, high density lipoprotein cholesterol, LDL-Chol, triglycerides and apo B levels genetic variability, d) the lipoproteine lipase (LPL) gene was responsible for 6.5% of the triglycerides variability, e) the apo E and LPL 447 polymorphisms influence in conjunction lipid parameters. These preliminary results on effects and combination effects of polymorphic genes show the interest of a multilocus approach. We have used in a subgroup of 416 individuals of a familial cohort (Stanislas Cohort) a prototype assay that genotypes a panel of 35 polymorphic sites on 15 candidate genes of Cardiovascular diseases. Each sample is amplified by two multiplex polymerase chain reactions, then hybridized to an array of immobilized, oligonucleotide probes. The frequencies of the rare alleles were in agreement with those reported by others in caucasian populations. The realisation of this multiplex assay in the 1,006 families of the Stanislas Cohort, which is underway, will allow us a better understanding of the inter-individual variability of lipids and will contribute to the determination of the genetic susceptibility of one's individual to cardiovascular risk.

Tinman function is essential for vertebrate heart development: elimination of cardiac differentiation by dominant inhibitory mutants of the tinman-related genes, XNkx2-3 and XNkx2-5.

In Drosophila, the tinman gene is absolutely required for development of the dorsal vessel, the insect equivalent of the heart. In vertebrates, the tinman gene is represented by a small family of tinman-related sequences, some of which are expressed during embryonic heart development. At present however, the precise importance of this gene family for vertebrate heart development is unclear. Using the Xenopus embryo, we have employed a dominant inhibitory strategy to interfere with the function of the endogenous tinman-related genes. In these experiments, suppression of tinman gene function can result in the complete elimination of myocardial gene expression and the absence of cell movements associated with embryonic heart development. This inhibition can be rescued by expression of wild-type tinman sequences. These experiments indicate that function of tinman family genes is essential for development of the vertebrate heart.

A multilocus genotyping assay for cardiovascular disease.

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.

Homeobox genes in cardiovascular development.

As summarized earlier, a surprisingly large number of different homeobox genes are expressed in the developing heart. Some are clearly important, as demonstrated by mouse gene ablation studies. For example, knockout of Nkx2-5 or Hoxa-3 function is embryonic lethal due to defects in cardiovascular development. However, gene ablation studies indicate that other homeobox genes that show cardiovascular expression are either not required for heart development or their function is effectively complemented by a redundant gene activity. Given the number of closely related homeobox genes that are expressed in the heart (and the rate at which new genes are being discovered), this is very likely to be the case for at least some homeobox gene activities. At present little is known of the precise mechanism of action of homeobox genes in embryonic development. This statement applies to homeobox genes in general, not just to genes involved in cardiovascular development. There is a popular view that homeobox genes are master regulators that control expression of a large number of downstream genes. In at least some cases, e.g., the eyeless gene of Drosophila (Holder et al., 1995), homeobox genes appear to be capable of activating and maintaining a very complex developmental program. Significantly, the eyeless gene is able to initiate eye development at numerous ectopic locations. Increasing evidence, however, suggests that genes of this type may be rather rare. Certainly there is no evidence to date that any of the homeobox genes expressed in the heart are able to initiate the complete heart development pathway. This is probably best understood in the case of the tinman gene in Drosophila, which, although absolutely required for heart development, is not capable of initiating the cardiac development pathway in ectopic locations (Bodmer, 1993). This conclusion is supported by studies of the vertebrate tinman-related gene Nkx2-5. Gene ablation studies show that Nkx2-5 is essential for correct cardiac development (Lyons et al., 1995) but is not able to initiate the regulatory pathway leading to cardiac development when expressed ectopically (Cleaver et al., 1996; Chen and Fishman, 1996). If most homeodomain proteins are not direct regulators of a differentiation pathway, what is their role during organogenesis? The cardiovascular homeobox gene about which most is known at the mechanistic level is gax (Smith et al., 1997). A number of experiments indicate that the Gax protein is involved in the regulation of cell proliferation and that it interacts with components of the cell cycle regulation machinery. Indeed, over recent years, the idea that at least some homeobox genes play their role in organogenesis through regulation of proliferation has been developed in some detail by Duboule (1995). Further evidence that this mechanism of homeobox activity is important, especially during organogenesis, comes from studies of the Hox11 homeobox gene, which is absolutely required for development of the spleen in mouse (Roberts et al., 1994). Studies indicate that Hox11 is able to interact with at least two different protein phosphatases, PP2A and PP1, which in turn, are involved in cell cycle regulation (Kawabe et al., 1997). It is quite clear that research in future years will need to focus on the precise mode of action of the different homeodomain proteins if we are to understand their role in the development of the cardiovascular system.

Remarkable sequence conservation of transcripts encoding amphibian and mammalian homologues of quaking, a KH domain RNA-binding protein.

Mutations in the mouse quaking locus can result in two different types of developmental phenotypes: (1) a deficiency of myelin in the central nervous system that is accompanied by a characteristic tremor, or (2) embryonic lethality around day 9 of gestation. A quaking candidate gene (qkI) that encodes a KH motif protein has recently been identified. We have isolated and characterized cDNAs encoding the Xenopus quaking homologue (Xqua) and also assembled an almost complete human quaking sequence from expressed sequence tags. Sequence comparisons show that the amphibian and mammalian quaking transcripts exhibit striking conservation, both within the coding region and, unexpectedly, in the 3' UTR. Two Xqua transcripts 5 kb and 5.5 kb in length are differentially expressed in the Xenopus embryo, with the 5 kb transcript being detected as early as the gastrula stage of development. Using an in vitro assay, we have demonstrated RNA-binding activity for quaking protein encoded by the 5 kb transcript. Overall, the high sequence conservation of quaking sequences suggests an important conserved function in vertebrate development, probably in the regulation of RNA metabolism.

Xbap, a vertebrate gene related to bagpipe, is expressed in developing craniofacial structures and in anterior gut muscle.

The Drosophila bagpipe (bap) gene is involved in the specification of the musculature of the embryonic midgut. We report the isolation and characterization of a Xenopus sequence, Xbap, which is closely related to bap. Xbap is also expressed in the developing musculature of the midgut, suggesting that this developmental role of bagpipe is evolutionarily conserved. However, a second, novel role in development is suggested by the observation that Xbap is also expressed in a region of the developing facial cartilage. Using a combination of cartilage staining and comparison to the goosecoid head expression pattern, we show that Xbap expression marks the precursors to the basihyobranchial, palatoquadrate, and possibly Meckel's cartilages. This vertebrate bagpipe sequence therefore is expressed in both mesodermally and neural crest-derived tissues.

Post-amplification primer extension of heat-denatured AmpliType PCR products: effects on typing results.

Alleles of the HLA, DQA1, LDLR, GYPA, HGBB, D7S8 and GC loci, which are amplified using the AmpliType(R) PM PCR Reaction Mix and Primer Set, can be detected using sequence-specific oligonucleotide probes immobilized on a nylon membrane strip. Using reagents supplied in AmpliType PCR Amplification and Typing Kits, patterns of blue dots corresponding to particular alleles are visualized on the DNA probe strips. Frequently, the correct interpretation of typing results is dependent not only on the presence of probe signals but also on their relative intensities. The relative probe signal intensities obtained from an undegraded DNA sample extracted from a single individual will be different from those obtained from degraded DNA and from samples containing DNA from more than one source. Because probe signal intensity is an essential consideration for interpretation, factors that can influence it need to be identified. Clearly, the time and temperature of the assay steps and the salt concentration in the typing solutions can affect probe signal intensity. Also, if heat-denatured PCR products are allowed to cool for several minutes, the strands will reanneal and become unavailable for binding to the probes immobilized on the strips. However, the selective loss of GC B and HLA DQA1 4.1 probe signals observed after shorter cooling times cannot be explained by these factors. We demonstrate that following heat denaturation of PM PCR products there is sufficient residual Taq DNA polymerase activity to extend primers as the solution cools and that this primer extension occurs at a more rapid rate than PCR product reannealing. Primer extension across probe binding sites will prevent hybridization of the PCR product to complementary probes on the strip. The extent of signal reduction is dependent on the position of the probe binding site relative to the 3' ends of the primers and on the strand to which the probe is complementary. We recommend a simple modification to the AmpliType typing protocol to ensure all probe binding sites will be available for hybridization to PM and HLA DQA1 DNA probe strips.

Integrating family and culture into medicine: a family systems block rotation.

BACKGROUND: Family-oriented care and sociocultural awareness are core competency objectives in behavioral science education. Nevertheless, teaching "family" and "culture" in ways that are meaningful and useful to residents can be a challenging task for family medicine educators. METHODS: We describe a block rotation that integrates family systems theory, family-oriented skills, and sociocultural awareness into residency education. Several objectives and teaching methods are presented in detail. RESULTS: Resident evaluations completed one year after graduation indicated that these skills were useful in practice. All respondents recommended continuation of the block rotation in the curriculum. CONCLUSION: The success of the rotation is due to a highly interactive small-group format, an emphasis on practical skills, and a multidisciplinary teaching approach. Advanced scheduling of faculty is necessary to ensure their uninterrupted participation in the rotation.