A Multilocus Genotyping Assay for Cardiovascular Disease

Cheng, Suzanne; Pallaud, Céline; Grow, Michael; Scharf, Stephen J.; Erlich, Henry A.; Klitz, William; Pullinger, Clive R.; Malloy, Mary J.; Kane, John P.; Siest, Gérard; Visvikis, Sophie

doi:10.1515/cclm.1998.096

Cited by 48 publications

(35 citation statements)

References 44 publications

(6 reference statements)

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“…The negative impact of both sampling and measurement errors is lessened. Logical first applications of the method proposed here would be fine mapping of candidate chromosomal regions already identified by family linkage or other studies and the testing of candidate genes chosen for their potential functional relationship to a disease (Cheng et al 1998). The advantages of low cost and effort, once regional SNPs are identified, are attractive in this context.…”

Section: Discussionmentioning

confidence: 99%

High-Throughput SNP Allele-Frequency Determination in Pooled DNA Samples by Kinetic PCR

Germer

Holland²,

Higuchi³

2000

Genome Res.

396

295

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We have developed an accurate, yet inexpensive and high-throughput, method for determining the allele frequency of biallelic polymorphisms in pools of DNA samples. The assay combines kinetic (real-time quantitative) PCR with allele-specific amplification and requires no post-PCR processing. The relative amounts of each allele in a sample are quantified. This is performed by dividing equal aliquots of the pooled DNA between two separate PCR reactions, each of which contains a primer pair specific to one or the other allelic SNP variant. For pools with equal amounts of the two alleles, the two amplifications should reach a detectable level of fluorescence at the same cycle number. For pools that contain unequal ratios of the two alleles, the difference in cycle number between the two amplification reactions can be used to calculate the relative allele amounts. We demonstrate the accuracy and reliability of the assay on samples with known predetermined SNP allele frequencies from 5% to 95%, including pools of both human and mouse DNAs using eight different SNPs altogether. The accuracy of measuring known allele frequencies is very high, with the strength of correlation between measured and known frequencies having an r 2 = 0.997. The loss of sensitivity as a result of measurement error is typically minimal, compared with that due to sampling error alone, for population samples up to 1000. We believe that by providing a means for SNP genotyping up to thousands of samples simultaneously, inexpensively, and reproducibly, this method is a powerful strategy for detecting meaningful polymorphic differences in candidate gene association studies and genome-wide linkage disequilibrium scans.

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Section: Discussionmentioning

confidence: 99%

High-Throughput SNP Allele-Frequency Determination in Pooled DNA Samples by Kinetic PCR

Germer

Holland²,

Higuchi³

2000

Genome Res.

396

295

View full text Add to dashboard Cite

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“…The genotyping of individuals for the TNF-a À308G/A, TNF-a À238G/A, IL-6 À174 G/C and IL-6 À572 G/C polymorphisms was performed using a multiplex assay, as described previously. 24 …”

Section: Blood Sample and Analysismentioning

confidence: 99%

Biological variations, genetic polymorphisms and familial resemblance of TNF-α and IL-6 concentrations: STANISLAS cohort

et al. 2004

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Cytokines are involved in the development of several inflammatory diseases and atherosclerosis. Their variations in healthy individuals are not well defined. The aims of this study were: firstly, to identify factors affecting biological variation of interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-a); secondly, to study their family resemblance; and thirdly, to evaluate the effect of two TNF-a (À308G/A and À238G/A) and two IL-6 polymorphisms (174G/C and À572G/C) on their corresponding circulating levels. A total of 171 healthy families selected from the STANISLAS cohort were studied. Age was negatively related to TNFa concentrations in offspring only (both sons and daughters). Additionally, IL-6 and TNF-a levels were differently influenced by gender, white blood cells, tobacco consumption, and HDL-cholesterol level. A weak significant familial resemblance for TNF-a concentration was observed in siblings only. There was no significant familial resemblance for IL-6 levels. The TNF-a À308A allele was associated with decreased TNFa concentrations in both offspring aged less than 18 and males without overweight (BMIo25 kg/m 2 ). Fathers carrying the IL-6 À174CC genotype had higher IL-6 levels than those with the IL-6 À174G allele. Parents with the IL-6 À572GG genotype had higher IL-6 concentrations than the C allele carriers. In this sample of healthy families, plasma levels of IL-6 and TNF-a were differently affected by biological parameters including age, gender and smoking, and the impact of their respective polymorphisms was influenced by gender, age and BMI.

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“…Genotyping was accomplished using multiplex polymerase chain reaction and immobilized probe-based assays for markers of cardiovascular disease, immune response, and inflammation, essentially as described elsewhere, but modified to also include genotyping capability for the P12A substitution (Roche Molecular Systems). 1,3,8,9 To confirm genotype assignments, the polymerase chain reaction procedure was performed in replicate on all samples, and scoring was carried out by 2 independent observers. Disagreements (Ͻ2%) were resolved by an additional joint reading and, where necessary, by repeat genotyping reaction.…”

Section: Genotypingmentioning

confidence: 99%

Alanine for Proline Substitution in the Peroxisome Proliferator–Activated Receptor Gamma-2 (PPARG2) Gene and the Risk of Incident Myocardial Infarction

Ridker

Cook

Cheng

et al. 2003

ATVB

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Objective-Recent studies have implicated the potential importance of peroxisome proliferator-activated receptors as a molecular mechanism involved in atherothrombosis. A common alanine (A) for proline (P) substitution at codon 12 in the peroxisome proliferator activated receptor gamma-2 gene (PPARG2) has been associated with reduced risk of developing type 2 diabetes mellitus. Because diabetes and atherothrombosis share common antecedents, we sought evidence that this polymorphism might also be associated with reduced risk of myocardial infarction. Methods and Results-Using DNA samples collected at baseline in a prospective cohort of 14 916 initially healthy American men, we evaluated a P12A polymorphism in the PPARG2 among 523 individuals who subsequently developed myocardial infarction and among 2092 individuals who remained free of reported cardiovascular disease over a mean follow-up period of 13.2 years. As hypothesized, presence of the A12 allele was associated with significantly reduced risk of myocardial infarction (odds ratio in an age-and smoking-adjusted dominant model of inheritance, 0.77; 95% CI, 0.60 to 0.98; Pϭ0.034). This protective effect remained statistically significant in analyses controlling for traditional cardiovascular risk factors, was present among nondiabetic study participants, was observed to be of similar magnitude in analyses assuming codominant or dominant modes of inheritance, and was seen in fully adjusted post hoc analyses in which we limited our control group to those individuals specifically matched to myocardial infarction cases (OR, 0.71; 95% CI, 0.53 to 0.96; Pϭ0.024). Key Words: genetics Ⅲ epidemiology Ⅲ myocardial infarction Ⅲ risk prediction Ⅲ polymorphism R ecent data suggest that a common alanine for proline substitution at codon 12 (P12A) in the peroxisome proliferator-activated receptor gamma-2 gene (PPARG2) is associated with reduced incidence of type 2 diabetes. [1][2][3][4] This observation is of considerable interest because patients with diabetes have accelerated atherosclerosis and insulin resistance is now recognized as a risk factor for myocardial infarction (MI). Recent experimental work has implicated that PPARG may act directly on local vasculature in several critical aspects of atherothrombosis, including lipid metabolism, foam cell responses, and inflammation, additionally suggesting that PPARG may be an important determinant of gene expression during atherogenesis. 5,6 We therefore hypothesized that the P12A polymorphism in the PPARG2 might also be a determinant of incident myocardial infarction (MI). Conclusions-In Methods Patient Selection and Clinical InvestigationWe sought evidence of association between a common P12A polymorphism in the PPARG2 and risk of incident MI as part of an ongoing genetic epidemiology evaluation of incident cardiovascular events being performed within the Physicians' Health Study. 7 In brief, of 22 071 American men aged 40 to 84 years who were free of prior MI, stroke, transient ischemic attack, and cancer, 14 916 provide...

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A Multilocus Genotyping Assay for Cardiovascular Disease

Cited by 48 publications

References 44 publications

High-Throughput SNP Allele-Frequency Determination in Pooled DNA Samples by Kinetic PCR

High-Throughput SNP Allele-Frequency Determination in Pooled DNA Samples by Kinetic PCR

Biological variations, genetic polymorphisms and familial resemblance of TNF-α and IL-6 concentrations: STANISLAS cohort

Alanine for Proline Substitution in the Peroxisome Proliferator–Activated Receptor Gamma-2 (PPARG2) Gene and the Risk of Incident Myocardial Infarction

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