Related Articles

AIM: To investigate effects of iron on oxidative stress, heme oxygenase-1 (HMOX1) and hepatitis C viral (HCV) expression in human hepatoma cells stably expressing HCV proteins.

METHODS: Effects of iron on oxidative stress, HMOX1, and HCV expression were assessed in CON1 cells. Measurements included mRNA by quantitative reverse transcription-polymerase chain reaction, and protein levels by Western blots.

RESULTS: Iron, in the form of ferric nitrilotriacetate, increased oxidative stress and up-regulated HMOX1 gene expression. Iron did not affect mRNA or protein levels of Bach1, a repressor of HMOX1. Silencing the up-regulation of HMOX1 nuclear factor-erythroid 2-related factor 2 (Nrf2) by Nrf2-siRNA decreased FeNTA-mediated up-regulation of HMOX1 mRNA levels. These iron effects were completely blocked by deferoxamine (DFO). Iron also significantly decreased levels of HCV core mRNA and protein by 80%-90%, nonstructural 5A mRNA by 90% and protein by about 50% in the Con1 full length HCV replicon cells, whereas DFO increased them.

CONCLUSION: Excess iron up-regulates HMOX1 and down-regulates HCV gene expression in hepatoma cells. This probably mitigates liver injury caused by combined iron overload and HCV infection.

Objective

To examine whether human endogenous retrovirus K10 is associated with autoimmune rheumatic disease.

Design

A novel multiplex reverse transcription polymerase chain reaction (RT‐PCR) system was developed to investigate HERV‐K10 mRNA expression in patients with rheumatoid arthritis.

Methods

40 patients with rheumatoid arthritis, 17 with osteoarthritis, and 27 healthy individuals were recruited and total RNA was extracted from peripheral blood mononuclear cells (PBMCs) and analysed using multiplex RT‐PCR for the level of HERV‐K10 gag mRNA expression. Southern blot and DNA sequencing confirmed the authenticity of the PCR products.

Results

Using the histidyl tRNA synthetase (HtRNAS) gene as a housekeeping gene in the optimised multiplex RT‐PCR, a significantly higher level of HERV‐K10 gag mRNA expression was found in rheumatoid arthritis than in osteoarthritis (p = 0.01) or in the healthy controls (p = 0.02).

Conclusion

There is enhanced mRNA expression of the HERV‐K10 gag region in rheumatoid arthritis compared with osteoarthritis or healthy controls. This could contribute to the pathogenesis of rheumatoid arthritis.

Background

The association of the deletion in GSTT1 and GSTM1 genes with coronary artery disease (CAD) among smokers is controversial. In addition, no such investigation has previously been conducted among Arabs.

Methods

We genotyped 1054 CAD patients and 762 controls for GSTT1 and GSTM1 deletion by multiplex polymerase chain reaction. Both CAD and controls were Saudi Arabs.

Results

In the control group (n = 762), 82.3% had the T wild M wildgenotype, 9% had the Twild M null, 2.4% had the Tnull M wild and 6.3% had the Tnull M null genotype. Among the CAD group (n = 1054), 29.5% had the Twild M wild genotype, 26.6% (p < .001) had the Twild M null, 8.3% (p < .001) had the Tnull M wild and 35.6% (p < .001) had the Tnull M null genotype, indicating a significant association of the Twild M null, Tnull M wild and Tnull M null genotypes with CAD. Univariate analysis also showed that smoking, age, hypercholesterolemia and hypertriglyceridemia, diabetes mellitus, family history of CAD, hypertension and obesity are all associated with CAD, whereas gender and myocardial infarction are not. Binary logistic regression for smoking and genotypes indicated that only M null and Tnullare interacting with smoking. However, further subgroup analysis stratifying the data by smoking status suggested that genotype-smoking interactions have no effect on the development of CAD.

Conclusion

GSTT1 and GSTM1 null-genotypes are risk factor for CAD independent of genotype-smoking interaction.

Background/Aims

BALB/c mice with a homozygous deficiency in the Tgfb1 gene are a model of fulminant autoimmune hepatitis (AIH), spontaneously and rapidly developing Th1-mediated IFN-γ-dependent necroinflammatory liver disease. We sought to understand the molecular basis for fulminant Th1 liver disease and the specific role of the Ifng gene.

Methods

Global gene expression in livers from BALB/c Tgfb1−/− mice with and without an intact Ifng gene was assessed by microarray analysis. Expression patterns were confirmed by quantitative reverse transcriptase-polymerase chain reaction. Gene ontology clustering analysis was performed to identify altered pathways. The contributions of Ifng to altered expression pathways were quantified.

Results

Over 100 genes were strongly (> 10-fold) upregulated, most encoding proteins involved in immune function/response. Chemokines were the most prominently upregulated group, with eight chemokine genes upregulated > 10-fold. Ifng was necessary for the upregulation of CXC chemokines gene, but not of CC chemokine genes. By quantitative analysis, Ifng’s role in liver gene upregulation varied greatly among overexpressed genes.

Conclusions

Gene expression changes indicate a particularly important and heretofore unappreciated role for chemokines in fulminant AIH. Ifng has an important role in expression of some but not all genes. Ifng is dichotomous in the regulation of distinct chemokine subfamilies: specifically, Ifng is critical for overexpression of specific CXCL genes but dispensable for overexpression of specific CCL genes. These results provide a clearer understanding of the role of Ifng in the molecular basis of necroinflammatory liver disease.

The implications of the methylene tetrahydrofolate reductase (MTHFR) gene and the level of homocysteine in the pathogenesis of coronary artery disease (CAD) have been extensively studied in various ethnic groups. Our aim was to discover the association of MTHFR (C677T) polymorphism and homocysteine level with CAD in north Indian subjects. The study group consisted of 329 angiographically proven CAD patients, and 331 age and sex matched healthy individuals as controls. MTHFR (C677T) gene polymorphism was detected based on the polymerase chain reaction and restriction digestion with HinfI. Total homocysteine plasma concentration was measured using immunoassay. T allele frequency was found to be significantly higher in patients than in the control group. We found significantly elevated levels of mean homocysteine in the patient group when compared to the control group (p = 0.00). Traditional risk factors such as diabetes, hypertension, smoking habits, a positive family history and lipid profiles (triglyceride, total cholesterol, HDL-cholesterol, LDL-cholesterol, VLDL-cholesterol), were found significantly associated through univariate analysis. Furthermore, multivariable logistics regression analysis revealed that CAD is significantly and variably associated with diabetes, hypertension, smoking, triglycerides and HDL-cholesterol. Our findings showed that MTHFR C677T polymorphism and homocysteine levels were associated with coronary artery disease in the selected population.

Background

Real-time reverse transcriptase quantitative polymerase chain reaction (real-time RTqPCR) is a technique used to measure mRNA species copy number as a way to determine key genes involved in different biological processes. However, the expression level of these key genes may vary among tissues or cells not only as a consequence of differential expression but also due to different factors, including choice of reference genes to normalize the expression levels of the target genes; thus the selection of reference genes is critical for expression studies. For this purpose, ten candidate reference genes were investigated in bovine muscular tissue.

Results

The value of stability of ten candidate reference genes included in three groups was estimated: the so called 'classical housekeeping' genes (18S, GAPDH and ACTB), a second set of genes used in expression studies conducted on other tissues (B2M, RPII, UBC and HMBS) and a third set of novel genes (SF3A1, EEF1A2 and CASC3). Three different statistical algorithms were used to rank the genes by their stability measures as produced by geNorm, NormFinder and Bestkeeper. The three methods tend to agree on the most stably expressed genes and the least in muscular tissue. EEF1A2 and HMBS followed by SF3A1, ACTB, and CASC3 can be considered as stable reference genes, and B2M, RPII, UBC and GAPDH would not be appropriate. Although the rRNA-18S stability measure seems to be within the range of acceptance, its use is not recommended because its synthesis regulation is not representative of mRNA levels.

Conclusion

Based on geNorm algorithm, we propose the use of three genes SF3A1, EEF1A2 and HMBS as references for normalization of real-time RTqPCR in muscle expression studies.

Background

For accuracy of quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), normalisation with suitable reference genes is required. To date, no reference genes have been validated for expression studies of bronchoalveolar (BAL) cells. The aims of this study were to identify gene(s) with stable mRNA expression in BAL cells irrespective of gender, smoking, BAL cellular composition, lung pathology, treatment; and to assess the influence of reference genes on target gene expression data.

Results

The mRNA expression of ten housekeeping genes (ACTB, ARF1, CANX, G6PD, GAPDH, GPS1, GNB2L1, PSMB2, PSMD2, RPL32) was investigated by qRT-PCR in BAL cells from 71 subjects across a spectrum of lung diseases. The analyses were validated in an independent BAL cohort from 63 sarcoidosis patients and 17 control subjects. A second derivative method was used to calculate expression values (CTt); an equivalence test, applets BestKeeper, geNorm and NormFinder were applied to investigate gene expression stability. Of the investigated genes, PSMB2 (CTt ± SD, 23.66 ± 0.86) and RPL32 (18.65 ± 0.92) were the most stable; both were constantly expressed in BAL samples from parallel investigated cohorts irrespective of evaluated variables. Finally, to demonstrate effect of traditional (ACTB/GAPDH) and novel (PSMB2/RPL32) reference genes as denominators, expression of two cytokines known associated with sarcoidosis was investigated in sarcoid BAL cells. While normalization with PSMB2/RPL32 resulted in elevated IFNG mRNA expression (p = 0.004); no change was observed using GAPDH/ACTB (p > 0.05). CCL2 mRNA up-regulation was observed only when PSMB2/RPL32 were used as denominators (p < 0.03).

Conclusion

PSMB2 and RPL32 are, therefore, suitable reference genes to normalize qRT-PCR in BAL cells in sarcoidosis, and other interstitial lung disease.

The stimulation of the human umbilical vein endothelial cell (HUVEC) with recombinant human monocyte-derived colony-stimulating factor (MCSF) increased the gene expression of monocyte chemotactic protein (MCP-1). Northern blot analysis indicated that 50 U/ml of MCSF is the optimal concentration for this effect. The elevation of MCP-1 mRNA started as early as 1 h after stimulation and was maintained for at least 8 h. An increased MCP-1 level in MCSF-treated HUVEC was also demonstrated at the protein level by immunocytochemical staining using a polyclonal MCP-1-specific antibody. HUVEC activated by 50 U/ml of MCSF for 5 h showed a stronger immunofluorescence staining than control cells. Micropipette separation of THP-1 monocytes from HUVEC showed that the activation of both THP-1 and endothelium by MCSF led to an increase in the separation force by more than three times (36.2 +/- 6.7 x 10(-4) vs. 9.6 +/- 3.6 x 10(-4) dyn). An increased adhesiveness was also observed after MCSF activation of peripheral blood monocytes and HUVEC (16.7 +/- 2.7 x 10(-4) vs. 5.2 +/- 0.9 x 10(-4) dyn). The increased adhesive force in both systems was blocked by the use of anti-MCP-1 (5.5 +/- 0.8 x 10(-4) and 6.8 +/- 1.1 x 10(-4) dyn). Similar results were obtained in experiments in which only HUVEC, but not monocytes, were activated by MCSF. This increased adhesion of untreated monocytes to MCSF-activated HUVEC was also blocked by the addition of anti-MCP-1. In contrast, experiments in which only THP-1 or peripheral blood monocytes, but not HUVEC, were treated with MCSF did not show a significant increase of adhesion between these cells. These results indicate that MCSF augments monocyte-endothelium interaction primarily by its action on the endothelial cell and that this function is probably mediated through an increased expression of MCP-1. The MCSF/MCP-1-dependent adhesive mechanism might be operative in the arterial wall in vivo to lead to the trapping of the infiltrated monocyte-macrophage in the subendothelial space during atherogenesis.

Images

Background

Quantitative real-time polymerase chain reaction (RT-qPCR) is valuable for studying the molecular events underlying physiological and behavioral phenomena. Normalization of real-time PCR data is critical for a reliable mRNA quantification. Here we identify reference genes to be utilized in RT-qPCR experiments to normalize and monitor the expression of target genes in the brain of the cephalopod mollusc Octopus vulgaris, an invertebrate. Such an approach is novel for this taxon and of advantage in future experiments given the complexity of the behavioral repertoire of this species when compared with its relatively simple neural organization.

Results

We chose 16S, and 18S rRNA, actB, EEF1A, tubA and ubi as candidate reference genes (housekeeping genes, HKG). The expression of 16S and 18S was highly variable and did not meet the requirements of candidate HKG. The expression of the other genes was almost stable and uniform among samples. We analyzed the expression of HKG into two different set of animals using tissues taken from the central nervous system (brain parts) and mantle (here considered as control tissue) by BestKeeper, geNorm and NormFinder. We found that HKG expressions differed considerably with respect to brain area and octopus samples in an HKG-specific manner. However, when the mantle is treated as control tissue and the entire central nervous system is considered, NormFinder revealed tubA and ubi as the most suitable HKG pair. These two genes were utilized to evaluate the relative expression of the genes FoxP, creb, dat and TH in O. vulgaris.

Conclusion

We analyzed the expression profiles of some genes here identified for O. vulgaris by applying RT-qPCR analysis for the first time in cephalopods. We validated candidate reference genes and found the expression of ubi and tubA to be the most appropriate to evaluate the expression of target genes in the brain of different octopuses. Our results also underline the importance of choosing a proper normalization strategy when analyzing gene expression by qPCR taking into appropriate account the experimental setting and variability of the sample of animals (and tissues), thus providing a set of HGK which expression appears to be unaffected by the experimental factor(s).

Background

Persistent residual immune activation and lipid dysmetabolism are characteristics of HIV positive patients receiving an highly active antiretroviral therapy (HAART). Nuclear Receptors are transcription factors involved in the regulation of immune and metabolic functions through the modulation of gene transcription. The objective of the present study was to investigate for the relative abundance of members of the nuclear receptor family in monocytic cells isolated from HIV positive patients treated or not treated with HAART.

Methods

Monocytes isolated from peripheral blood mononuclear cells (PBMC) were used for analysis of the relative mRNA expressions of FXR, PXR, LXR, VDR, RARα, RXR, PPARα, PPARβ, PPARγ and GR by Real-Time polymerase chain reaction (PCR). The expression of a selected subset of inflammatory and metabolic genes MCP-1, ICAM-1, CD36 and ABCA1 was also measured.

Results

Monocytes isolated from HIV infected patients expressed an altered pattern of nuclear receptors characterized by a profound reduction in the expressions of FXR, PXR, PPARα, GR, RARα and RXR. Of interest, the deregulated expression of nuclear receptors was not restored under HAART and was linked to an altered expression of genes which supports both an immune activation and altered lipid metabolism in monocytes.

Conclusions

Altered expression of genes mediating reciprocal regulation of lipid metabolism and immune function in monocytes occurs in HIV. The present findings provide a mechanistic explanation for immune activation and lipid dysmetabolism occurring in HIV infected patients and could lead to the identification of novel potential therapeutic targets.

Background

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated transcription factor, which regulates gene expression of the key proteins involved in lipid metabolism, vascular inflammation, and proliferation. PPARγ may contribute to attenuating atherogenesis and postangioplasty restenosis. PPARγ C161→T substitution is associated with a reduced risk of coronary artery disease (CAD). Whether or not the gene substitution alters the risk of CAD in type 2 diabetes mellitus (T2DM) patients remains unclear.

Methods

A total of 556 unrelated subjects from a Chinese Han population, including 89 healthy subjects, 78 CAD patients, 86 T2DM patients, and 303 CAD combined with T2DM patients, were recruited to enroll in this study. PPARγC161→T gene polymorphism was determined by polymerase chain reaction and restriction fragment length polymorphisms. Plasma levels of lipoproteins, apolipoproteins, glucose, and insulin were measured by ELISA or radioimmunoassay (RIA). The coronary artery lesions were evaluated by coronary angiography.

Results

The frequency of the 161T allele in CAD, T2DM, and CAD combined with T2DM patients was similar to that observed in the healthy control group. However, in CAD combined with T2DM patients, the group with angiographically documented moderate stenoses had a higher frequency of the 161T allele in comparison to the group with severe stenoses (P < 0.05). Moreover, in CAD with T2DM patients, the triglyceride levels and apoB in CC homozygote carriers were significantly higher than those in "T" allele carriers.

Conclusions

PPARγC161→T genotypes weren't significantly associated with the risk of CAD, but were markedly correlated with severity of disease vessels in patients with CAD and T2DM. Furthermore, PPARγC161→T substitution was associated with an altered adipose, but not glucose metabolism. These results indicate that the PPARγ C161→T polymorphism may reduce the risk of severe atherogenesis by modulation of adipose metabolism, especially triglycerides and apoB, in Chinese patients with CAD and T2DM.

BACKGROUND:

Single nucleotide polymorphisms in the 5,10-methylenetetrahydrofolate reductase (MTHFR), vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), monocyte chemoattractant protein-1 (MCP-1) and apolipoprotein E (ApoE) genes appear to be a genetic risk factor for atherosclerosis. Common carotid intima-media thickness (cIMT) provides information on the severity of atherosclerosis.

OBJECTIVE:

To investigate the relationship between cIMT and gene polymorphisms associated with atherosclerosis in Turkish patients with coronary artery disease (CAD).

METHODS:

Sixty-two patients with angiographically diagnosed stable CAD were divided into two groups according to their cIMT values (group 1: n=35, cIMT of 1 mm or greater; group 2: n=27, cIMT of less than 1 mm). MTHFR 677 C/T, VEGF–460 C/T, eNOS 894 G/T, MCP-1–2518 A/G and ApoE (E2, E3 and E4) gene polymorphisms (where A is adenine, C is cytosine, G is guanine and T is thymine) were analyzed by polymerase chain reaction and restriction fragment length polymorphism. Evaluations of cardiovascular risk factors and coronary atherosclerotic lesions were performed in all patients. Serum homocysteine and high-sensitivity C-reactive protein were measured and compared between the two groups.

RESULTS:

Serum high-sensitivity C-reactive protein (P=0.04) and homocysteine (P=0.006) levels were higher in group 1 than in group 2. The ratio of multivessel CAD and previous myocardial infarction was significantly higher in group 1 than in group 2 (P=0.014). In the study population, no significant difference in cIMT was observed according to the polymorphisms studied. Only hyperhomocysteinemia (OR 1.17 [95% CI 1.01 to 1.35], P=0.033) and previous myocardial infarction (OR 3.76 [95% CI 1.10 to 12.81], P=0.034) maintained a significant correlation with cIMT on multiple logistic regression analysis.

CONCLUSION:

cIMT is increased in patients with hyperhomocysteinemia, inflammation and extended CAD. MTHFR 677 C/T, VEGF–460 C/T, eNOS 894 G/T, MCP-1–2518 A/G and ApoE single nucleotide polymorphisms were not associated with increased cIMT.

BACKGROUND AND OBJECTIVES:

Endo-derived nitric oxide (NO) is synthesized from L-arginine by endothelial nitric oxide synthase (NOS3). Since reduced NO synthesis in endothelial cells has been implicated in the development of coronary atherosclerosis, we investigated the association of NOS3 gene polymorphisms and coronary artery disease (CAD) in an Iranian population.

SUBJECTS AND METHODS:

We studied the NOS3 gene Glu298Asp polymorphism in 241 CAD patients with positive coronary angiograms (i.e., >50% stenosis affecting at least one coronary vessel) in Shahid Rajaee Heart Hospital and 261 control subjects without a history of symptomatic CAD. The NOS3 gene polymorphism was analyzed by polymerase chain reaction and restriction fragment length polymorphism. Lipid profile and other risk factors were also determined.

RESULTS:

The genotype frequencies of Glu298Asp polymorphism for Glu/Glu, Glu/Asp, and Asp/Asp were 61.3%, 32.2%, and 6.5%, respectively, in control subjects, and 46.5%, 42.7%, and 10.8% in CAD patients, respectively. The genotype frequencies differed significantly between the two groups (P=.003). The frequencies of the Asp alleles were 32.2% and 22.6% for CAD patients and control subjects, respectively; the difference between the two groups was statistically significant (P=.001; odds ratio=1.6). Plasma lipids, except HDL-C, were also significantly increased in the CAD groups.

CONCLUSION:

These results suggest that CAD is associated with Glu298Asp polymorphism of the NOS3 gene in our population and that this polymorphism is an independent risk factor for CAD.

Aim

The goal of this investigation was to determine whether epigenetic modifications in the IFNG promoter are associated with an increase of IFNG transcription in different stages of periodontal diseases.

Materials and Methods

DNA was extracted from gingival biopsy samples collected from 47 total sites from 47 different subjects: 23 periodontally healthy sites, 12 experimentally induced gingivitis sites and 12 chronic periodontitis sites. Levels of DNA methylation within the IFNG promoter containing six CpG dinucleotides were determined using pyrosequencing technology. Interferon gamma mRNA expression was analysed by quantitative polymerase chain reactions using isolated RNA from part of the biological samples mentioned above.

Results

The methylation level of all six analysed CpG sites within the IFNG promoter region in the periodontitis biopsies {52% [interquartile range, IQR (43.8%, 63%)]} was significantly lower than periodontally healthy samples {62% [IQR (51.3%, 74%)], p =0.007} and gingivitis biopsies {63% [IQR (55%, 74%)], p =0.02}. The transcriptional level of IFNG in periodontitis biopsies was 1.96-fold and significantly higher than tissues with periodontal health (p =0.04). Although the mRNA level from experimental gingivitis samples exhibited an 8.5-fold increase as compared with periodontally healthy samples, no significant methylation difference was observed in experimental gingivitis sample.

Conclusions

A hypomethylation profile within IFNG promoter region is related to an increase of IFNG transcription present in the chronic periodontitis biopsies, while such an increase of IFNG in experimentally induced gingivitis seems independent of promoter methylation alteration.

The cad operon encodes lysine decarboxylase and a protein homologous to amino acid antiporters. These two genes are induced under conditions of low pH, anaerobiosis, and excess lysine. The upstream regulatory region of the cad operon has been cloned into lacZ expression vectors for analysis of the sequences involved in these responses. Deletion analysis of the upstream region and cloning of various fragments to make cadA::lacZ or cadB::lacZ protein fusions or operon fusions showed that cadA was translated more efficiently than cadB and localized the pH-responsive site to a region near an upstream EcoRV site. Construction of defined end points by polymerase chain reaction further localized the left end of the regulatory site. The presence of short fragments bearing the regulatory region on high-copy-number plasmids greatly reduced expression from the chromosomal cad operon, suggesting that titration of an essential activator protein was occurring. With nonoptimal polymerase chain reaction conditions, a set of single point mutants were made in the upstream regulatory region. Certain of these altered regulatory regions were unable to compete for the regulatory factor in vivo. The locations of these essential bases indicate that a sequence near the EcoRV site is very important for the activator-DNA interaction. In vivo methylation experiments were conducted with cells grown at pH 5.5 or at pH 8, and a difference in protection was observed at specific G residues in and around the region defined as important in pH regulation by the mutation studies. This work defines essential sequences for acid induction of this system involved in neutralization of extracellular acid.

Images

BACKGROUND AND OBJECTIVES:

The initial step in atherosclerosis is the adhesion of leukocytes to activated endothelial cells mediated by intercellular adhesion molecule-1 (ICAM-1). This study aimed to investigate the association of K469E polymorphism of the ICAM-1 gene and soluble ICAM-1 (sICAM-1) serum level with coronary heart disease (CHD) in Egyptian subjects.

PATIENTS AND METHODS:

Using a case-control design, we studied 100 patients with CHD, including 73 patients with acute myocardial infarction (MI) and 27 with unstable angina (UA). The control group consisted of 50 healthy subjects with normal left ventricular function. All participants were genotyped for the ICAM-1 polymorphism by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Serum sICAM-1 was measured by enzyme-linked immunoassay (ELISA).

RESULTS:

In CHD patients, the frequencies of K genotype (KK and EK) were significantly higher when compared to controls (P<.001) and were associated with an increased risk of disease development (OR=3.8, 95% CI: 1.7 to 8.5; P=.001). K genotype frequencies in patients with MI showed no significant difference when compared to patients with UA (P= .121). Serum sICAM-1 levels were comparable between CHD patients and controls (P= .37) and between MI and UA patients (P=.23). There were no significant differences in sICAM-1 levels among patients with different genotypes (P=.532). Men presented with higher sICAM-1 levels than women (P=.004).

CONCLUSION:

ICAM-1 gene polymorphism in codon 469 is associated with a risk for CHD development in Egyptian subjects. Serum sICAM-1 is not influenced by this polymorphism and is not necessarily elevated in CHD.

A single-nucleotide promoter region polymorphism (-108C/T) of the paraoxonase (PON1) gene had been suggested to influence an individual's susceptibility to coronary artery disease (CAD). No data is available on this polymorphism from India. One hundred seventy-eight healthy individuals and 204 angiographically proven CAD patients were recruited to get baseline data on the frequency distribution of the -108C/T polymorphism in normal people of Asian Indian ethnicity and its relation with the risk of CAD. Polymerase chain reaction followed by restriction fragment length analysis was used as the method for genotyping. Blood samples were used for DNA isolation. In the normal subjects, the genotypes were distributed as CT (43.26%) > CC (30.34%), >TT (26.4%). The allele frequency of the C allele was 0.52, and that of the T allele was 0.48. The patients showed a similar pattern, but the TT genotype was about two times more frequent in the controls than in patients. Odds ratios for developing CAD for individuals with CT, TT, and CT + TT genotypes were 0.89 (0.50–1.59), 0.56 (0.27–1.08), and 0.76 (0.44–1.29), respectively (at 95% confidence interval), when compared to CC homozygous people (age- and sex-adjusted, p = 0.114, all genotypes compared). This suggested a trend for the T allele as protective against CAD. This first report on the frequency distribution of the -108C/T polymorphism in people of Asian Indian ethnicity suggests that the normal distribution is similar to that observed for the Chinese, Japanese, and Latino people, but the disease association is unique. The TT genotype and the T allele which are widely found associated with the risk of CAD showed a protective trend in this study.

Abstract

Coronary artery disease (CAD) is a multifactorial disease where genetic and environmental factors interact in complex ways to cause the disease. Heat shock protein genes are involved in the progress of CAD. This implies that genetic variants of Hsp70–2 genes might contribute to the development of the CAD.

Aim of study

The aim of this study was to characterize statistical correlation of linkage between lipid profiles, polymorphism PstI site of Hsp70–2 gene and CAD.

Patients and methods

This study was carried out on Tunisian patients with CAD recruited from Hospital of Fattouma Bourguiba of Monastir-Tunisia. Polymerase chain reaction and restriction enzymes were used to determine the genotypic distributions in 252 unrelated patients and 151 healthy control subjects. Further, ApoA-I and ApoB as well as the serum total of cholesterol, HDL, triglyceride, and hs-CRP levels were measured.

Results

We showed a decreased level of ApoA-I, whereas the levels of each of ApoB and hs-CRP were increased in patients with CAD compared with control group. In addition our studies of a polymorphic PstI site of Hsp70-2 gene at position 1267 of the Hsp70–2 gene have revealed that the allelic frequency of P2 was significantly more frequent in CAD patients than controls group (p=0.007, OR=1.495). The genotypic distribution showed a high incidence of P2/P2 genotype in CAD patients (0.190) compared to healthy control (0.009) with reach significant difference (p=0.006). The P2 carriers showed a significantly increased of Total-Cholesterol (CT) and C-reactive protein (hs-CRP) levels in CAD patients (p=0.008 and p=0.018, respectively).

Conclusion

The high incidence of P2-Hsp70-2 genotype in CAD patients and the significantly association of P2/P2 genotype with elevated Total Cholesterol and hs-CRP levels, supported that P2–Hsp70–2 genotype has susceptibility implication in CAD and could increased the risk of CAD in Tunisian population.

Virtual slides

The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1118340895703689

Background

Reference genes, which are often referred to as housekeeping genes are frequently used to normalize mRNA levels between different samples in quantitative reverse transcription polymerase chain reaction (qRT-PCR). The selection of reference genes is critical for gene expression studies because the expression of these genes may vary among tissues or cells and may change under certain circumstances. Here, a systematic evaluation of six putative reference genes for gene expression studies in human hepatocellular carcinoma (HCC) is presented.

Methods

Six genes, beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyl-transferase 1 (HPRT1), succinate dehydrogenase complex, subunit A (SDHA) and ubiquitin C (UBC), with distinct functional characteristics and expression patterns were evaluated by qRT-PCR. Inhibitory substances in RNA samples were quantitatively assessed and controlled using an external RNA control. The stability of selected reference genes was analyzed using both geNorm and NormFinder software.

Results

HMBS and GAPDH were identified as the optimal reference genes for normalizing gene expression data between paired tumoral and adjacent non-tumoral tissues derived from patients with HCC. HMBS, GAPDH and UBC were identified to be suitable for the normalization of gene expression data among tumor tissues; whereas the combination of HMBS, B2M, SDHA and GAPDH was suitable for normalizing gene expression data among five liver cancer cell lines, namely Hep3B, HepG2, HuH7, SK-HEP-1 and SNU-182. The determined gene stability was increased after exclusion of RNA samples containing relatively higher inhibitory substances.

Conclusion

Of six genes studied, HMBS was found to be the single best reference gene for gene expression studies in HCC. The appropriate choice of combination of more than one reference gene to improve qRT-PCR accuracy depends on the kind of liver tissues or cells under investigation. Quantitative assessment and control of qRT-PCR inhibitors using an external RNA control can reduce the variation of qRT-PCR assay and facilitate the evaluation of gene stability. Our results may facilitate the choice of reference genes for expression studies in HCC.

Glutathione S-transferase (GST) plays a key role in the detoxification of xenobiotic atherogen generated by smoking. To analyze the effect of GSTM1/T1 gene polymorphisms on the development of smoking-related coronary artery disease (CAD), 775 Korean patients who underwent coronary angiography were enrolled. The subjects were classified by luminal diameter stenosis into group A (>50%), B (20-50%), or C (<20%). GSTM1 and GSTT1 gene polymorphisms were analyzed using multiplex polymerase chain reaction (PCR) for GSTM1/T1 genes and CYP1A1 gene for internal control. Of 775 subjects, 403 patients belonged to group A. They had higher risk factors for CAD than group B (N=260) and group C (N=112). The genotype frequencies of null GSTM1 and GSTT1 showed no significant differences among 3 groups. Considering the effect of GSTM1 gene polymorphisms on the smoking-related CAD, smokers with GSTM1 null genotype had more increased risk for CAD than non-smoker with GSTM1 positive genotype (odds ratios [OR], 2.07, confidence interval [CI], 1.06-4.07). Also the effect of GSTT1 gene polymorphism on smoking-related CAD showed the same tendency as GSTM1 gene (OR, 2.00, CI, 1.05-3.84). This effect of GSTM1/T1 null genotype on smoking-related CAD was augmented when both gene polymorphisms were considered simultaneously (OR, 2.76, CI, 1.17-6.52). We concluded that GSTM1/T1 null genotype contributed to the pathogenesis of smoking-related CAD to some degree.

Background & objectives:

Hyperhomocysteinaemia (HCA) either due to mutation of MTHFR gene or deficiency of vitamin B12 and folic acid, has been reported as a risk factor for coronary artery disease (CAD). The present study was aimed to determine plasma homocysteine (Hcy) levels and to evaluate MTHFR C677T gene polymorphism as risk factors for CAD, and to study the role of Hcy in conjunction with a few other risk factors of CAD in young Indians. The effect of vitamin B12 and folic acid supplements on the raised plasma Hcy levels in patients of CAD was also assessed.

Methods:

The present study included 199 consecutive angiography confirmed CAD patients, <45 yr of age, without any other known pro- coagulant state and 200 age- and sex-matched healthy controls. Fasting blood samples were collected in EDTA and plasma Hcy was estimated by ELISA test and the MTHFR C677T polymorphism detection was carried out by PCR-RFLP method.

Results:

Significant difference (P<0.001) was found between mean fasting levels of plasma Hcy in cases (22.14 ± 10.62 μmol/l) and controls (17.38 ± 8.46 μmol/l) with an Odds ratio as 1.93 (95% CI, 1.27-2.94). Levels of cholesterol, LDL, and triglycerides were significantly (P<0.001) higher in cases compared with controls.

Interpretation & conclusions:

Our study showed significant correlation between hyperhomocysteinaemia and coronary artery disease. Multivariate analysis by logistic regression of the various risk factors of CAD, found high levels of Hcy, cholesterol, LDL and low levels of HDL and smoking as independent predictors of CAD when all other factors were controlled. Significant post-treatment decrease found in HCA was easily modifiable by vitamin intervention irrespective to their CT or TT genotype of C677T MTHFR gene. Further studies to look at the plasma levels of folate and cobalamines and their association with Hcy are required to be done.

Background

Hyperhomocysteinemia as a consequence of the MTHFR 677 C > T variant is associated with cardiovascular disease and stroke. Another factor that can potentially contribute to these disorders is a depleted nitric oxide level, which can be due to the presence of eNOS +894 G > T and eNOS −786 T > C variants that make an individual more susceptible to endothelial dysfunction. A number of genotyping methods have been developed to investigate these variants. However, simultaneous detection methods using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis are still lacking. In this study, a novel multiplex PCR-RFLP method for the simultaneous detection of MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants was developed. A total of 114 healthy Malay subjects were recruited. The MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants were genotyped using the novel multiplex PCR-RFLP and confirmed by DNA sequencing as well as snpBLAST. Allele frequencies of MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C were calculated using the Hardy Weinberg equation.

Methods

The 114 healthy volunteers were recruited for this study, and their DNA was extracted. Primer pair was designed using Primer 3 Software version 0.4.0 and validated against the BLAST database. The primer specificity, functionality and annealing temperature were tested using uniplex PCR methods that were later combined into a single multiplex PCR. Restriction Fragment Length Polymorphism (RFLP) was performed in three separate tubes followed by agarose gel electrophoresis. PCR product residual was purified and sent for DNA sequencing.

Results

The allele frequencies for MTHFR 677 C > T were 0.89 (C allele) and 0.11 (T allele); for eNOS +894 G > T, the allele frequencies were 0.58 (G allele) and 0.43 (T allele); and for eNOS −786 T > C, the allele frequencies were 0.87 (T allele) and 0.13 (C allele).

Conclusions

Our PCR-RFLP method is a simple, cost-effective and time-saving method. It can be used to successfully genotype subjects for the MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants simultaneously with 100% concordance from DNA sequencing data. This method can be routinely used for rapid investigation of the MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C variants.

Background

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful method for the analysis of gene expression. Target gene expression levels are usually normalized to a consistently expressed reference gene also known as internal standard, in the same sample. However, much effort has not been expended thus far in the search for reference genes suitable for the study of stomach cancer using RT-qPCR, although selection of optimal reference genes is critical for interpretation of results.

Methods

We assessed the suitability of six possible reference genes, beta-actin (ACTB), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyl transferase 1 (HPRT1), beta-2-microglobulin (B2M), ribosomal subunit L29 (RPL29) and 18S ribosomal RNA (18S rRNA) in 20 normal and tumor stomach tissue pairs of stomach cancer patients and 6 stomach cancer cell lines, by RT-qPCR. Employing expression stability analyses using NormFinder and geNorm algorithms we determined the order of performance of these reference genes and their variation values.

Results

This RT-qPCR study showed that there are statistically significant (p < 0.05) differences in the expression levels of HPRT1 and 18S rRNA in 'normal-' versus 'tumor stomach tissues'. The stability analyses by geNorm suggest B2M-GAPDH, as best reference gene combination for 'stomach cancer cell lines'; RPL29-HPRT1, for 'all stomach tissues'; and ACTB-18S rRNA, for 'all stomach cell lines and tissues'. NormFinder also identified B2M as the best reference gene for 'stomach cancer cell lines', RPL29-B2M for 'all stomach tissues', and 18S rRNA-ACTB for 'all stomach cell lines and tissues'. The comparisons of normalized expression of the target gene, GPNMB, showed different interpretation of target gene expression depend on best single reference gene or combination.

Conclusion

This study validated RPL29 and RPL29-B2M as the best single reference genes and combination, for RT-qPCR analysis of 'all stomach tissues', and B2M and B2M-GAPDH as the best single reference gene and combination, for 'stomach cancer cell lines'. Use of these validated reference genes should provide more exact interpretation of differential gene expressions at transcription level in stomach cancer.

The genes encoding β-actin (ACTB in human or Actb in mouse) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH in human or Gapdh in mouse) are the two most commonly used references for sample normalization in determination of the mRNA level of interested genes by reverse transcription (RT) and ensuing polymerase chain reactions (PCR). In this study, bioinformatic analyses revealed that the ACTB, Actb, GAPDH and Gapdh had 64, 69, 67 and 197 pseudogenes (PGs), respectively, in the corresponding genome. Most of these PGs are intronless and similar in size to the authentic mRNA. Alignment of several PGs of these genes with the corresponding mRNA reveals that they are highly homologous. In contrast, the hypoxanthine phosphoribosyltransferase-1 gene (HPRT1 in human or Hprt in mouse) only had 3 or 1 PG, respectively, and the mRNA has unique regions for primer design. PCR with cDNA or genomic DNA (gDNA) as templates revealed that our HPRT1, Hprt and GAPDH primers were specific, whereas our ACTB and Actb primers were not specific enough both vertically (within the cDNA) and horizontally (compared cDNA with gDNA). No primers could be designed for the Gapdh that would not mis-prime PGs. Since most of the genome is transcribed, we suggest to peers to forgo ACTB (Actb) and GAPDH (Dapdh) as references in RT-PCR and, if there is no surrogate, to use our primers with extra caution. We also propose a standard operation procedure in which design of primers for RT-PCR starts from avoiding mis-priming PGs and all primers need be tested for specificity with both cDNA and gDNA.

Because many species-specific phenotypic differences are assumed to be caused by differential regulation of gene expression, many recent investigations have focused on measuring transcript abundance. Despite the availability of high-throughput platforms, quantitative real-time polymerase chain reaction (RT-QPCR) is often the method of choice because of its low cost and wider dynamic range. However, the accuracy of this technique heavily relies on the use of multiple valid control genes for normalization. We created a pipeline for choosing genes potentially useful as RT-QPCR control genes for measuring expression between human and chimpanzee samples across multiple tissues, using published microarrays and a measure of tissue-specificity. We identified 13 genes from the pipeline and from commonly used control genes: ACTB, USP49, ARGHGEF2, GSK3A, TBP, SDHA, EIF2B2, GPDH, YWHAZ, HPTR1, RPL13A, HMBS, and EEF2. We then tested these candidate genes and validated their expression stability across species. We established the rank order of the most preferable set of genes for single and combined tissues. Our results suggest that for at least three tissues (cerebral cortex, liver, and skeletal muscle), EIF2B2, EEF2, HMBS, and SDHA are useful genes for normalizing human and chimpanzee expression using RT-QPCR. Interestingly, other commonly used control genes, including TBP, GAPDH, and, especially ACTB do not perform as well. This pipeline could be easily adapted to other species for which expression data exist, providing taxonomically appropriate control genes for comparisons of gene expression among species.