Pyrosequencing Analysis of Thrombosis-Associated Risk Markers

Holmberg, Kristina; Persson, Marie-Louise; Uhlén, Mathias; Odeberg, Jacob

doi:10.1373/clinchem.2005.049932

Cited by 15 publications

(13 citation statements)

References 13 publications

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“…All loci were amplified in an outer 10-plex PCR followed by an inner 10-plex PCR for genotyping with PrASE and also 10 inner simplex PCR reactions for genotyping with Pyrosequencing. Pyrosequencing was performed as previously described [17]. A 99.8 % concordance was achieved between the two assays.…”

Section: Resultsmentioning

confidence: 99%

“…DNA was extracted from blood from unrelated individuals of Caucasian/Scandinavian origin (from a cohort of patients presenting with symptoms of acute chest pain) [17]. The patients were included in the Carlscrona Heart Attack Prognosis Study approved by the ethics committee at the University of Lund, Sweden in compliance with the Declaration of Helsinki.…”

Section: Methodsmentioning

confidence: 99%

“…Ten polymorphic positions previously studied with Pyrosequencing [17] for their suggested association as prothrombotic genetic variations [18-20] were used (Table 1, amplicon GC contents of 38 to 69%). These were analyzed on genomic DNA from unrelated DNA samples of Caucasian/Scandinavian origin from a cohort of patients presenting with symptoms of acute chest pain [17]. A total of 4420 genotypes were scored by each method and accordingly this study offers a thorough characterization of a microarray-based technique in terms of accuracy, robustness and variability.…”

Section: Introductionmentioning

confidence: 99%

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Comparison of PrASE and Pyrosequencing for SNP Genotyping

et al. 2006

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Background: There is an imperative need for SNP genotyping technologies that are cost-effective per sample with retained high accuracy, throughput and flexibility. We have developed a microarray-based technique and compared it to Pyrosequencing. In the protease-mediated allelespecific extension (PrASE), the protease constrains the elongation reaction and thus prevents incorrect nucleotide incorporation to mismatched 3'-termini primers.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Comparison of PrASE and Pyrosequencing for SNP Genotyping

et al. 2006

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“…Allele-specific loading of Pol II was evaluated by Pyrosequencing™ as described [13]. Briefly, DNA was amplified using primer pairs with one biotinylated 5' end (Table S3) [14]. The amplification products were captured on streptavidin coated sepharose beads (Amersham Biosciences AB, Uppsala, Sweden), denatured and then washed.…”

Section: Methodsmentioning

confidence: 99%

A Common Polymorphism in the Promoter Region of the TNFSF4 Gene Is Associated with Lower Allele-Specific Expression and Risk of Myocardial Infarction

et al. 2011

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BackgroundThe TNFSF4/TNFRSF4 system, along with several other receptor-ligand pairs, is involved in the recruitment and activation of T-cells and is therefore tentatively implicated in atherosclerosis and acute coronary syndromes. We have previously shown that genetic variants in TNFSF4 are associated with myocardial infarction (MI) in women. This prompted functional studies of TNFSF4 expression.Methods and ResultsBased on a screening of the TNFSF4 genomic region, a promoter polymorphism (rs45454293) and a haplotype were identified, conceivably involved in gene regulation. The rs45454293T-allele, in agreement with the linked rs3850641G-allele, proved to be associated with increased risk of MI in women. Haplotype-specific chromatin immunoprecipitation of activated polymerase II, as a measure of transcriptional activity in vivo, suggested that the haplotype including the rs45454293 and rs3850641 polymorphisms is functionally important, the rs45454293T- and rs3850641G-alleles being associated with lower transcriptional activity in cells heterozygous for both polymorphisms. The functional role of rs45454293 on transcriptional levels of TNFSF4 was clarified by luciferase reporter assays, where the rs45454293T-allele decreased gene expression when compared with the rs45454293C-allele, while the rs3850641 SNP did not have any effect on TNFSF4 promoter activity. Electromobility shift assay showed that the rs45454293 polymorphism, but not rs3850641, affects the binding of nuclear factors, thus suggesting that the lower transcriptional activity is attributed to binding of one or more transcriptional repressor(s) to the T-allele.ConclusionsOur data indicate that the TNFSF4 rs45454293T-allele is associated with lower TNFSF4 expression and increased risk of MI.

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“…A pyrosequencing-based genotype platform that analyzes common prothrombotic, hemostatic, and treatment-modulating mutations may represent an optimized diagnostic strategy for analysis ( Holmberg et al, 2005 ), with potential application in both epidemiologic studies and clinical diagnosis ( Mooser et al, 2003 ).…”

Section: Emerging Platform For Hemostasis and Thrombosis Researchmentioning

confidence: 99%