Tuba Gunel

Serum microRNA expression in pregnancies with preeclampsia

Preeclampsia continues to be a mortal disease of pregnant women throughout the world. Recently, geneticists, allied with obstetricians, have opened new frontiers. MicroRNAs (miRNAs) are members of a class of small, noncoding RNA molecules. They are critical posttranscriptional regulators of gene expression. We extracted circulating miRNA from maternal plasma and quantified mir-152 and mir-210. We found up-regulated miR-210 levels as well as down-regulated mir-152 levels in preeclampsia patients.We propose that detection of increased mir-210 levels in maternal serum could be used to improve prediction methods for noninvasive prenatal diagnosis of preeclampsia.

Clinical evaluations of cell-free fetal DNA quantities in pre-eclamptic pregnancies

Quantitative changes of cell‐free fetal DNA in maternal plasma as an indicator for impending pre‐eclampsia was reported in different studies. Cell‐free fetal nucleic acids can be detected in maternal circulation during pregnancy. Our aim was to determine the higher rate of fetal DNA levels in maternal blood in pre‐eclampsia compared to normal pregnancies and the clinical use of real‐time polymerase chain reaction (PCR) in the Turkish population as a marker.

Metabolic Engineering for Production of Geranylgeranyl Pyrophosphate Synthase in Non-Carotenogenic Yeast Schizosaccharomyces Pombe

ABSTRACT Geranylgeranyl pyrophosphate synthase (GGPPS) is a key enzyme in carotenoid biosynthetic pathway, catalyzing the synthesis of its C20 precursor. The gene encoding GGPPS from bell pepper (Capsicum annuum) has been cloned in Schizosaccharomyces pombe and its heterologous expression analyzed in this model organism for eukaryotes. We aim to partially direct the ergosterol pathway to carotenoid biosynthesis in this yeast. This yeast is not able to synthesize carotenoids naturally, but it produces farnesyl pyrophosphate (FPP), an intermediate metabolite for both ergosterol and geranylgeranyl pyrophosphate (GGPP) biosynthesis. S. pombe transformants carrying the pepper GGPPS cDNA were named KTG. In one of the transformant (KTG1), heterologous expression of GGPPS gene has been analyzed. The GGPPS gene was shown by dot and Northern hybridization to be successfully transcribed. When water soluble proteins isolated from the transformant cells have been analyzed on SDS-denaturating gels, a ~37 kDaprotein band has been detected which was not found in the host. In order to understand whether this heterologous protein in KTG1 transformant is an active GGPPS or not, changes in ergosterol level have been analyzed by HPLC. 50–60% decrease in ergosterol content was found in the KTG1 transformant when compared to the host. These results suggest that production of GGPP derivatives can now be attempted, for instance carotenoids, through heterologous expression of carotenoid biosynthetic genes in a non-carotenogenic yeast, S. pombe.

Investigation of Preeclampsia Using Raman Spectroscopy

Preeclampsia is associated with increased perinatal morbidity and mortality. There have been numerous efforts to determine preeclampsia biomarkers by means of biophysical, biochemical, and spectroscopic methods. In this study, the preeclampsia and control groups were compared via band component analysis and multivariate analysis using Raman spectroscopy as an alternative technique. The Raman spectra of serum samples were taken from nine preeclamptic, ten healthy pregnant women. The Band component analysis and principal component analysis-linear discriminant analysis were applied to all spectra after a sensitive preprocess step. Using linear discriminant analysis, it was found that Raman spectroscopy has a sensitivity of 78% and a specificity of 90% for the diagnosis of preeclampsia. Via the band component analysis, a significant difference in the spectra of preeclamptic patients was observed when compared to the control group. 19 Raman bands exhibited significant differences in intensity, while 11 of them decreased and eight of them increased. This difference seen in vibrational bands may be used in further studies to clarify the pathophysiology of preeclampsia.

Effect of angiotensin I-converting enzyme and α-actinin-3 gene polymorphisms on sport performance

Genetic polymorphism is considered to be associated with human physical performance. The angiotensin I-converting enzyme insertion/deletion (ACE I/D) and the α-actinin-3 gene (ACTN3) R577X polymorphisms have been widely investigated for such associations, and functional ACE I/D and ACTN3 R577X polymorphisms have been associated with sprinter performance. The aim of this study was to determine the effect of these polymorphisms on sport performance among 37 elite athletes and 37 healthy controls. The ACE II genotype was identified in 32.43% of the control group and 8.11% of elite athletes, the DD genotype in 37.84% of the control group and 51.35% of the elite athletes, and the ID genotype in 29.73% of the control group and 40.54% of the elite athletes. With regard to the ACTN3 gene, the XX genotype, which confers an advantage for endurance activities, was identified in 10.81% of the control group and 35.14% of the elite athletes. The XX genotype was observed more frequently than the RR genotype (advantageous for sprinting), which was identified in 2.70% of the control group and 10.81% of elite athletes. The RX genotype (observed in 86.48% of the control group and in 54.05% of the elite athletes) was the most common genotype of the individuals in the present study. The study showed that ACTN3 and ACE gene polymorphisms have an effect on muscle power; however, larger studies are required.

Detection of fetal RHD pseudogene (RHDΨ) and hybrid RHD-CE-Ds from RHD-negative pregnant women with a free DNA fetal kit.

Hemolytic disease of the newborn is a clinical condition in which maternal and paternal Rh blood group antigens are incompatible and the mother is negative for the antigen whereas the father is positive. Analysis of fetal cells recovered from maternal plasma can provide a highly sensitive prenatal diagnosis. The fetal RHD gene in plasma DNA is detected by real-time PCR amplification of two different segments of the RHD gene (exons 7 and 10). Each amplicon is revealed with specific probes. We examined 40 female blood samples to verify the specificity of RHD exons (7 and 10) amplified by real-time PCR. Thirty fetuses were predicted to be RHD-positive based on analysis of plasma DNA. Seven fetuses were predicted to be RHD-negative. One fetus was negative for RHD on exon 10, and positive for RHD on exon 7 (early gestation age); two fetuses were RHD-negative on exon 7, and RHD-positive on exon 10 (RHD-CE-D(s) or RHDΨ), indicative of a maternal RHD allele. We conclude that it is necessary to analyze at least two exon regions in the RHD gene.

Comparison of real-time polymerase chain reaction assay methods for detection of RHD gene in amniotic fluid

Hemolytic disease of the newborn is the clinical condition in which Rh blood group antigens in couples are incompatible with each other and mother is negative for the antigen, whereas father is positive. Although RHD antigen encoded by RHD gene that is localized on chromosome 1 determines persons Rh genotyping, this incompatibility can lead to delivery as anemia, jaundiced, or dead in mothers uterus. In recent years, improvements have occurred in the prenatal diagnosis of Rh incompatibility. Quantitative real-time polymerase chain reaction (Real-time PCR) has been improved and determining rapidly, reliably, and sensitively has been possible. In this study, the determination of RHD genotyping was investigated using fetal DNA obtained from amniotic fluid and SYBR Green I and TaqMan probe methods were compared, and reliability in prenatal diagnosis of these methods was determined. We studied 35 pregnant women in the second trimester of pregnancy. “SYBR Green I” and “TaqMan” probes results for RHD gene of genomic DNA extracted from total 35 different amniotic fluid samples acquired from 10 RHD (-) and 25 pregnant women randomly were analyzed. DNA extracted from amniotic fluid was analyzed for RHD gene with real-time PCR and the results were then compared with the RHD fetal genotype determined on RHD phenotype of the red blood cells of the infants at birth. The results of RHD TaqMan probes PCR analysis of amniotic fluid DNA were completely concordant with the fetal blood group analysis after birth. Real-time PCR using the TaqMan probes has proven to be more sensitive, accurate, and specific for RHD gene than SYBR Green I method.

Large Scale Pre-Diagnosis of Toxoplasma Gondii DNA Genotyping by Real-Time PCR on Amniotic Fluid

ABSTRACT The antepartum and peripartum maternal infections cause great problems complicating pregnancy. The early diagnosis of the maternal infections causing different pathologies in the newborns is of great importance. For an appropiate judgement, the early diagnosis of Toxoplasma gondii infections acquired during pregnancy is critical for an effective prevention. The common characteristics of these infections are that they may cause abortion, still birth, prematurity, intrauterin growth retardation, congenital malformations, infection of the newborn or normal term live-births. We report here the development of real-time PCR-based assay for detection of T. gondii. Investigation of 300 specimens was carried out using B1 gene region specific primers and probes after the extraction of T. gondii DNA. T.gondii DNA, was found in 4 (1.3%) out of 300 specimens. DNA was not found in the speciemens of the remaining 296 patients. Real-time PCR analysis significantly improves the detection of T. gondii in amniotic fluid.

MicroRNA expression profiling in placenta and maternal plasma in early pregnancy loss

Early pregnancy loss (EPL), also termed early miscarriage, is determined as the unintentional expulsion of an embryo or fetus prior to the 12th week of gestation. EPL frequency is ~15% in pregnancies. Fetal development and growth is associate with placental function and vessel development; therefore, the placental genome would represent a useful miscarriage model for (epi)genetic and genomic studies. An important factor of placental development and function is epigenetic regulation of gene expression. microRNAs (miRNAs) are the primary epigenetic regulators which have an important role in placental development and function. In the present study, maternal plasma and villous tissue were collected from 16 EPL cases in 6th-8th gestational weeks (GWs) and 8 abortions (control group) in 6th-8th GWs. Detection of the differences in miRNA expression was performed using microarrays and dysregulated miRNAs were validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). miRNA microarray findings revealed that four miRNAs, including hsa-miRNA (miR)-125a-3p, hsa-miR-3663-3p, hsa-miR-423-5p and hsa-miR-575 were upregulated in tissue samples. In maternal plasma, two miRNAs (hsa-let-7c, hsa-miR-122) were upregulated and one miRNA (hsa-miR-135a) was downregulated. A total of 6 out of 7 dysregulated miRNAs were validated using RT-qPCR. The target genes of these dysregulated miRNAs were detected using the GeneSpring database. The aim of the present study was to detect dysregulated miRNAs in maternal plasma and villous cells and identify the target genes of dysregulated miRNAs and their associated pathways. The target gene analyses have revealed that the affected genes are primarily associated with cell migration, proliferation, implantation, adhesion, angiogenesis and differentiation and all are involved with EPL pathogenesis. Therefore, the present study may contribute to the understanding of the molecular mechanisms which lead to EPL.

Future Perspective of Preeclampsia by miRNA.

MicroRNAs (miRNAs) are single-stranded RNA (20-23 bp) molecules which regulate expression of proteincoding gene by promoting mRNA degradation and translational inhibition in mammalians. The latest miRBase release (v20, June 2013) contains 24 521 microRNA loci from 206 species which is processed to produce 30,424 mature microRNA products. miRNAs are involved in several mechanisms such as cardiovascular, central nervous system processes and cancer. miRNAs can be produced by human placenta and are significant for placental development. Recently, regulation of miRNA expression is not exactly understood in human placenta, however many research demonstrated that oxygen tension, signaling molecules, environmental toxin and epigenetic regulation are playing critical roles in placental development. Placental miRNAs that are used for prenatal diagnosis have been detected in maternal plasma in recent years. Some complications of pregnancy such as preeclampsia which is also regulated by miRNA could be diagnosed by using miRNA techniques. In preeclamptic blood and tissue samples, upor down-regulated miRNA levels can be observed. In this study, topics such as miRNA biogenesis and regulation in pregnancy, pregnancy associated disorders are referred and future perspective of prenatal diagnosis using miRNA as biomarkers.