INTRODUCTION: the FcgammaRIIa receptor is responsible for the clearance of large immune complexes and recently has been proved to be a C-reactive protein (CRP) receptor as well. A polymorphism in the corresponding FCG2RA gene resulting in an amino acid change (R131H) has been implicated, with conflicting results in the pathogenesis of various autoimmune or inflammatory disorders (e.g., atherosclerosis and coronary artery disease [CAD]). METHODS: we recently developed a real-time polymerase chain reaction and melting curve analysis method for the genotyping of the above polymorphism. We further looked at its validity with bioinformatics study and DNA sequencing. Then we genotyped 134 CAD patients and 45 angiographically normal controls and determined serum high-sensitivity CRP by nephelometry (Dade-Behring). Also, we used apparently healthy platelet donors (n = 206) as a larger control group. RESULTS: our method is accurate and devoid of problems with homologs and copy number variants. The need for reference materials is stressed. There were statistically significant differences (p < 0.05) between the CAD patients and each of the two other control groups, with the percentage of RR genotype rising from 6.5% and 11% in the control groups to an average of 19% in all CAD patients (17%, 24%, and 18.5% in stable angina, unstable angina, and myocardial infarction, respectively). In a logistic regression model that included known risk factors for CAD including CRP, the RR genotype remained a significant predictor for CAD (odds ratio: 6.3 [1.1-36.3]). Also after linear regression analysis, CRP levels were reduced in the RR carriers (vs. HH + HR), controlling for age, sex, and disease (marginal p = 0.07). CONCLUSIONS: with our accurate genotyping method, the RR genotype was correlated with atherothrombotic CAD events. The inverse correlation found between CRP levels and genotype supports the in vitro data of RR cells binding CRP stronger than HH

Somatic mutations in the PIK3CA gene have been discovered in many human cancers, and their presence correlates to therapy response. Three "hotspot" mutations within the PIK3CA gene are localized in exons 9 and 20. High-resolution melting analysis (HRMA) is a highly sensitive, robust, rapid, and cost-effective mutation analysis technique. We developed a novel methodology for the detection of hotspot mutations in exons 9 and 20 of the PIK3CA gene that is based on a combination of PCR and HRMA. The PIK3CA HRMA assay was evaluated by performing repeatability, sensitivity, and comparison with DNA sequencing studies and was further validated in 129 formalin-fixed paraffin-embedded breast tissue samples: 99 tumors, 20 noncancerous, and 10 fibroadenomas. The developed methodology was further applied in a selected group of 75 breast cancer patients who underwent Trastuzumab treatment. In sensitivity studies, the assay presented a capability to detect as low as 1% of mutated dsDNA in the presence of wtDNA for both exons. In the 99 tumor samples (validation group), 12/99 (12.1%) exon 9 mutations and 20/99 (20.2%) exon 20 mutations were found. No mutations were found in noncancerous tissues. In fibroadenomas, we report one PIK3CA mutation for the first time. In the selected group, 30/75 (40%) samples were detected as mutants. The PIK3CA HRMA assay is highly sensitive, reliable, cost-effective, and easy-to-perform, and therefore can be used as a screening test in a high-throughput pharmacodiagnostic setting

OBJECTIVES: 5382insC frameshift mutation along with 5331G>A (G1738R) missense mutation, both found in exon 20 of the BRCA1 gene, are relatively frequent among the Greek breast and ovarian cancer population (46%). Our goal was to develop a novel, reliable and rapid genotyping/scanning method for mutation detection of the exon 20 of the BRCA1 gene, using high-resolution melting curve analysis. DESIGN AND METHODS: The developed methodology was based on real-time PCR and high-resolution melting curve analysis in the presence of LCGreen I dye. Two amplicons on the exon 20 of BRCA1 gene were designed (157 bp and 100 bp), one flanking the exon's boundaries, and one embracing the 5382insC mutation. Our methodology was first optimized and validated by using genomic DNA samples with the 5382insC and 5331G>A (G1738R) mutations and wild-type. In total, the developed methodology was applied on 90 peripheral blood and 127 formalin-fixed paraffin-embedded breast tissue samples. RESULTS: Sensitivity studies with gDNA isolated from peripheral blood showed that mutated DNA could be reliably detected in the presence of wild-type DNA at 5% and 0.5% ratio with the larger and the smaller amplicon, respectively. By using the developed methodology we successfully identified 5382insC, 5331G>A and 5370C>T (R1751X) mutations, in genomic DNA isolated from peripheral blood samples and 5382insC mutation in two breast tumors, as verified by DNA sequencing. CONCLUSIONS: The combination of real-time PCR and high-resolution melting curve analysis provides a cost-efficient, simple and rapid approach to successfully scan exon 20 of BRCA1 gene for these clinically important and frequent mutations