Relative quantitation of cell-free fetal DNA in maternal plasma using autosomal DNA markers.

BACKGROUND: Cell-free fetal DNA in maternal plasma presents interest as it can possibly serve as a pregnancy associated biomarker. For its quantitation, we investigate the use of fluorescent polymerase chain reaction (PCR) amplification of short tandem repeats (STRs) in pregnancies irrespective of sex. METHODS: Artificially chimeric DNA samples were prepared so as to simulate cell-free fetal DNA in maternal plasma at different proportions. The samples were tested with fluorescent PCR amplification of informative STRs and the percentage of the population simulating fetal DNA was plotted against a relative ratio of the alleles detected in each sample. The graphs were used for the quantitation of cell-free fetal DNA in 50 maternal plasma samples. RESULTS: Detection of at least one paternally inherited fetal STR was possible in 46/50 pregnancies leading to the estimation of the relative percentage of cell-free fetal DNA. Four pregnancies failed to reveal any fetal allele which may reflect undetectable levels (<1%) of fetal DNA. Among pregnancies fetal DNA ranged from 0 to 20% (mean value of 7%). CONCLUSION: Our system can readily estimate the relative levels of cell-free fetal DNA in maternal circulation, normal gestations of either sex and can be exploited for estimating possible variations of this analyte between normal and pathological pregnancies.

Lack of association between the cholesteryl ester transfer protein gene--TaqIB polymorphism and coronary restenosis following percutaneous transluminal coronary angioplasty and stenting: a pilot study.

BACKGROUND: The most widely studied variation at the cholesteryl ester transfer protein (CETP) gene locus is a silent base change called the Thermobius aquaticus IB (TaqIB) polymorphism. TaqIB has been shown to affect levels/activity of CETP, plasma levels of high-density lipoprotein cholesterol (HDL-C), and to contribute to the risk of developing atherosclerosis and coronary heart disease (CHD). Ongoing studies are investigating possible associations between CETP gene polymorphisms and the development of coronary restenosis following percutaneous transluminal coronary angioplasty (PTCA) and stenting. METHODS AND RESULTS: The primary objective of the present study was to investigate the frequency of TaqIB-polymorphism, and a possible association with post-PTCA coronary restenosis, in 204 Greek patients who had undergone PTCA and stenting. As a secondary objective, the analysis was extended to explore possible interacting or additive effects by various CHD risk factors, and a deletion in the alpha(2B)-adrenergic receptor gene. The frequency of TaqIB was 54%, similar to the frequency of the polymorphism in a group of 35 healthy controls. CONCLUSIONS: The results from this study do not indicate that the TaqIB variation at the CETP gene locus is a significant predictor for assessing the risk of developing coronary restenosis following PTCA and stenting. This result was not affected when considering any one of the additionally studied factors.

Positive association between two polymorphic sites (+134 insA/delA and G198T) of the endothelin-1 gene and chronic obstructive pulmonary disease. A case-control study.

Endothelin-1 (ET-1) has been implicated in the pathogenesis of Chronic Obstructive Pulmonary Disease (COPD) for establishing an inflammatory loop in the respiratory mucosa that could become independent from the initial irritant factor. Common causes of COPD exacerbations are associated with elevated ET-1 sputum concentrations. Genetic variants of the ET-1 gene, that lead to elevated ET-1 peptide levels, have not been investigated in COPD. We performed a case control, genetic study to assess possible associations of two polymorphisms of the ET-1 gene, an adenine insertion (+134 insA/delA) and a guanine to thymine transversion (G198T) with the COPD phenotype and disease severity. The genotypes of 209 subjects, 107 COPD smokers (patients) and 102 non-COPD smokers (controls) were examined. Statistical analysis revealed that the 3A/4A and 4A/4A genotypes were more common (P<0.01) in patients. Moreover, a protective effect against COPD of the TT genotype (G198T) was exhibited. COPD smokers were carrying more frequently the GG genotype and less frequently the TT genotype (P=0.047). Diplotypic analysis revealed that subjects carrying the 3A3A;TT genotype had a lower risk of COPD development (P=0.027). Within the COPD patient group carriers of the GT genotype had more often mild or moderate COPD compared to patients carrying the GG genotype (P=0.004). Haplotypic distribution revealed that carriers of the 4A:T and 4A:G haplotypes were in increased risk of COPD development. Additionally, patients with the 3A:G haplotype were in increased risk of developing severe COPD, whereas patients with the 3A:T and 4A:T had most probably mild-moderate COPD.

Physiological levels of HBB transgene expression from S/MAR element-based replicating episomal vectors.

Replicating episomal vectors (REV) are in principle able to provide long-term transgene expression in the absence of integration into the target cell genome. The scaffold/matrix attachment region (S/MAR) located 5' of the human beta-interferon gene (IFNB1) has been shown to confer a stable episomal replication and retention function within plasmid vectors when stably transfected and selected in mammalian cells. The minimal requirement for the IFNB1 S/MAR to function in DNA replication and episomal retention is transcription through this element. We used the erythroid beta-globin locus control region-beta-globin gene (betaLCR-HBB) microlocus cassette as a model to assess tissue-specific expression from within an IFNB1 S/MAR-based plasmid REV. The betaLCR-HBB plus S/MAR combination constructs provided either high or low levels of transcription through the S/MAR element. Our results show that the betaLCR-HBB microlocus is able to reproducibly and stably express at full physiological levels on an episome copy number basis. In addition, our data show that even low levels of transcription from betaLCR-HBB through the S/MAR element are sufficient to allow efficient episomal replication and retention. These data provide the principles upon which generic and flexible expression cassette-S/MAR-based REVs can be designed for a wide range of applications.

Lack of association between alpha2B-adrenergic receptor polymorphism and risk of restenosis following coronary angioplasty and stent implantation--preliminary report.

BACKGROUND: A genetic association/prospective follow-up study was conducted to investigate whether genetic variation of the alpha(2B)-adrenergic receptor gene was associated with the risk of restenosis in 96 Greek coronary artery disease patients undergoing coronary angioplasty and stent implantation. METHODS: For comparison of genotype frequency, a control group of 83 asymptomatic individuals was also studied. The end-point of the current study was the incidence of restenosis at 7 months of clinical follow-up. RESULTS: The majority of patients (70/96) had the insertion/insertion genotype, fewer patients (23/96) had the insertion/deletion genotype and only 3/96 had the deletion/deletion genotype; overall the frequency distribution was not different from that of the control subjects. Restenosis occurred in 15 of the 96 patients. CONCLUSIONS: In the population studied, alpha(2B)-adrenoreceptor polymorphisms were not found to predispose patients to an increased incidence of restenosis. Nevertheless, these findings should be considered as preliminary, taking into account the small number of patients that were studied and the rarity of the deletion/deletion genotype.