Improved real-time RT-PCR assays of two colorectal cancer peripheral blood mRNA biomarkers: a pilot study

Background: efficient screening for detection of colorectal cancer [CRC] at earlier stages reduces its mortality. The purpose of this study was to investigate expression of carcinoembryonic antigen [CEA] and human telomerase reverse transcriptase [hTERT] mRNA in peripheral blood of CRC patients and to present strategies for early detection screen test

Methods: twenty seven patients in non-metastatic stage and 27 healthy individuals were studied. Expression of CEA, hTERT mRNA and 18srRNA [18s subunit of ribosomal RNA, as reference gene] were determined based on real-time RT-PCR on 3 [micro]g of total RNA from blood in 3 separate vials [1 [micro]g per vial]

Results: positive expression rate of CEA mRNA [78%] and hTERT mRNA [81%] were higher in patient group [P<0.001]. These rates were meaningfully higher than the results of individual vials containing only 1 [micro]g of total RNA. Difference between Ct values of markers with 18srRNA [[DELTA]Ct] was higher in healthy group than patient one. Therefore, a [DELTA]Ct cut-off value was determined for distinguishing between true- and false-positive results. Concurrent expression of both markers was found in 67% of the patients, which was higher than healthy cases [11%]. Combination of concurrent marker expression with cut-off point strategy increased specificity to 100%

Conclusion: these results showed that concurrent evaluation of marker expression and performing the test on 3 [micro]g of samples in 3 separate vials may increase specificity and sensitivity of real-time RT-PCR for early detection of non-metastatic CRC. However, more investigations with larger numbers of samples are needed to verify these results. Iran. Biomed. J. 17 [1]: 15-21, 2013

[Association of -971 G/A cholesteryl-ester transfer protein gene polymorphism with lipid profile in primary hyperlipidemia]

Coronary heart disease [CHD] is a leading cause of death worldwide and hypertriglyceridemia and hypercholesterolemia are major risk factors for the disease. Considering the role of hyperlipidemia as the underlying cause of cardiovascular mortalities and morbidities, and the limited and conflicting results of studies on CETP gene polymorphisms in Iran, we aimed to study -971 G/A polymorphism of cholesterol ester transfer protein gene in patients with primary hyperlipidemia. In this case-control study performed in Hamadan University of Medical Sciences [from May 2010 to April 2011], we recruited 200 patients with primary hyperlipidemia [total cholesterol > 250 mg/dl and/or triglyceride > 200 mg/dl] as the cases and 200 healthy individuals with normal cholesterol and triglyceride as the control group. Gene segments were replicated by polymerase chain reaction [PCR] and -971 G/A polymorphism genotypes were identified by RFLP technique. Subsequently, plasma CETP activity was measured enzymeatically by a kit in a fluorescence spectrometer. The allele and genotype frequencies were not significantly different [P > 0.05] between the two groups [in the control group: AA 24%, GA 47% and GG 28.5% and in the case group: AA 18%, GA 51% and GG 31%]. In the case group, homozygous individuals with A alleles [AA genotype] had greater cholesterol and HDL-c concentrations than those with other alleles [GG and GA]. In both cases and controls, individuals with AA genotype had lower CETP concentrations. We conclude that -971 G/A polymorphism in CETP gene promoter can affect lipid profile and alter CETP activity

[Association between cholestryl ester transfer protein D442G polymorphism on serum lipid levels and CETP activity in hypercholesterolemic patients]

Hypercholesterolemia is considered a major risk factor for pancreatitis, atherosclerosis and coronary heart disease. Cholesteryl ester transfer protein gene polymorphisms are known to be associated with changes in lipid levels. We investigated the association between a polymorphism in the CETP gene [D442G] with plasma lipid levels and CETP activity in patients with hypercholesterolemia. This case/control study that be done in Hamadan university of medical sciences [from October 2008 to September 2009], included 102 patients with hypercholesterolemia and 200 healthy individuals. Polymerase chain reaction and restriction fragment length polymorphisms were used to determine genotypic distribution and allelic frequencies of polymorphisms. The plasma CETP activity was measured by a kit in a fluorescence spectrometer. Lipid concentrations were measured by routine biochemical and enzymatic assays. Plasma cholesteryl ester transfer protein activity was significantly higher in the cases than the controls [P<0.05]. The genotypic and allelic frequencies for this polymorphism were not statistically different between the patients with hypercholesterolemia and the controls [in controls: DD 96%, DG 4%, GG 0% and in cases: DD 86%, DG 10%, GG 4%], [P>0.05]. Plasma HDL-C, LDL-C and TC were higher in both groups with GG and DG genotypes than with DD genotype, whereas serum CETP activity was lower in GG genotype compared with other genotypes [GD or DD], [P<0.05]. The results showed that D442G polymorphism of CETP gene was associated with changes in lipid profile and plasma CETP activity in the selected population and it might have a role in contributing to a genetic risk for developing coronary artery disease