PubMed Health⌕ Search

Biomedical subjects

Sinuhe Hahn

Publications and source records attributed to Sinuhe Hahn.

At least 19 recordsLinked to original sources
PubMed2007

Non-invasive prenatal detection of achondroplasia in size-fractionated cell-free DNA by MALDI-TOF MS assay.

Achondroplasia is the most common form of short-limbed dwarfism in humans and is caused by mutations in the FGFR3 gene. Currently, prenatal diagnosis of this disorder relies on invasive procedures. Recent studies have shown that fetal single gene point mutations could be detected in cell-free DNA (cf-DNA) from maternal plasma by either the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) assay with single allele base extension reaction (SABER) approach or the size fractionation of cf-DNA in maternal plasma. Here, we combined the two approaches to non-invasively examine the fetal G1138A mutation in maternal plasma. cf-DNA was extracted from maternal plasma samples obtained from two pregnant women at risk for achondroplasia. The fetal G1138A mutation was determined by the analysis of size-fractionated cf-DNA in maternal plasma using MALDI-TOF MS with SABER approach and homogenous MassEXTEND (hME) assay, respectively. The fetal G1138A mutation was detectable in the two achondroplasia-affected pregnancies by the analysis of cf-DNA in maternal plasma using MALDI-TOF MS. However, the size-fractionation approach led to a more precise detection of the fetal mutation in both analyses. This analysis would be suitable for non-invasive prenatal diagnosis of diseases caused by fetal single gene point mutations.

Achondroplasia↗
PubMed2006

Is the quantity of circulatory cell-free DNA in human plasma and serum samples associated with gender, age and frequency of blood donations?

Circulatory cell-free DNA (cf-DNA) is increased in a variety of clinical pathologic conditions; therefore, these markers could be widely used as markers for detecting and monitoring several disorders. To better understand the biology of this molecule, we analysed the relationship between the level of circulatory cf-DNA and physiological parameters such as gender, age and frequency of blood donations. Paired plasma and serum samples were obtained from 87 blood donors and 50 healthy adults who had never donated blood. Cf-DNA was extracted from plasma and serum samples using the MagNA Pure LC Instrument. Quantity determination of circulatory cf-DNA was performed by TaqMan real-time PCR for the ubiquitous GAPDH gene. Our data showed that the concentration of cf-DNA in serum was about eightfold higher than that in plasma. Regarding the level of these circulatory species, no significant differences were observed between the age-matched men and women and gender-matched, different-age cohorts, except in women who were older than 60 years of age. Frequent blood donations did not increase the circulatory species. Circulatory cf-DNA in plasma and serum samples is not correlated with human gender and human age except in women who are older than 60 years of age. Frequent blood donation did not affect the quantity of circulatory cf-DNA. The explanation for the latter most likely is the short half-life time of free fetal DNA in maternal circulation.

Adult↗
PubMed2006

Quantitative analysis of intact fetal cells in maternal plasma by real-time PCR.

OBJECTIVE: Fetal genetic materials in maternal circulation might be potentially used for non-invasive prenatal diagnosis (NIPD). In this study, we quantitatively analysed the intact fetal cells existing in maternal plasma, which would be a special method of NIPD. STUDY DESIGN: Eleven samples from preeclamptic patients, two samples from patients with RhD incompatibilities, and 30 samples from normal pregnancies as controls were collected. The intact cells in the plasma fraction were separated by centrifugation at high speed. The intact cell pellets were washed with PBS to remove any cell-free DNA. RESULTS: We were able to detect intact fetal cells in 3 out of 11 preeclamptic plasma samples from women carrying male fetuses, and from both plasma samples of RhD incompatibility affected pregnancies. However, intact fetal cells were not detected in all the 16 normal pregnancies with a male fetus, although cell-free fetal DNA was detected in all these cases. CONCLUSIONS: Intact fetal cells might be present in maternal plasma. However, the rarity of these cells limits their use for reliable, non-invasive prenatal diagnosis. The detection of such cells was successful in some preeclamptic and RhD incompatibility samples, not in the normal controls. Therefore, as opposed to free fetal DNA in maternal blood the use of intact fetal cells does not provide a reliable mode for NIPD.

Cell Separation↗
PubMed2006

In utero haematopoietic stem cell transplantation. Experiences in mice, sheep and humans.

Early prenatal diagnosis and in utero therapy of certain fetal diseases have the potential to reduce fetal morbidity and mortality. The intrauterine transplantation of stem cells provides in some instances a therapeutic option before definitive organ failure occurs. Clinical experiences show that certain diseases, such as immune deficiencies or inborn errors of metabolism, can be successfully treated using stem cells derived from bone marrow. However, a remaining problem is the low level of engraftment that can be achieved. Efforts are made in animal models to optimise the graft and study the recipient's microenvironment to increase long-term engraftment levels. Our experiments in mice show similar early homing of allogeneic and xenogeneic stem cells and reasonable early engraftment of allogeneic murine fetal liver cells (17.1% donor cells in peripheral blood 4 weeks after transplantation), whereas xenogeneic HSC are rapidly diminished due to missing self-renewal and low differentiation capacities in the host's microenvironment. Allogeneic murine fetal liver cells have very good longterm engraftment (49.9% donor cells in peripheral blood 16 weeks after transplantation). Compared to the rodents, the sheep model has the advantage of body size and gestation comparable to the human fetus. Here, ultrasound-guided injection techniques significantly decreased fetal loss rates. In contrast to the murine in utero model, the repopulation capacities of allogeneic ovine fetal liver cells are lower (0.112% donor cells in peripheral blood 3 weeks after transplantation). The effect of MHC on engraftment levels seems to be marginal, since no differences could be observed between autologous and allogeneic transplantation (0.117% donor cells vs 0.112% donor cells in peripheral blood 1 to 2 weeks after transplantation). Further research is needed to study optimal timing and graft composition as well as immunological aspects of in utero transplantation.

Animals↗
PubMed2006

Disturbances in placental immunology: ready for therapeutic interventions?

Recent studies have provided new insight into aberrations in the immunological interplay between mother and fetus and their potential role in the development of recurrent fetal loss and preeclampsia. The action of anti-phospholipid antibodies in recurrent fetal loss is now proposed to involve the complement system, neutrophil activation and the production of TNFalpha by immune bystander cells. A clear involvement of the immune system is emerging in preeclampsia, involving mainly the innate arm, especially neutrophils. The activation of peripheral neutrophils by placentally released inflammatory debris triggers the induction of neutrophil extracellular traps (NETs), which may lead to an occlusion of the intervillous space, thereby further promoting a condition of placental hypoxia. It has, hence, been suggested that new therapeutic strategies be developed, including the possible use of TNFalpha antagonists in cases of recurrent miscarriage. These strategies need to be addressed with caution due to the possible induction of fetal congenital abnormalities.

Abortion, Habitual↗
PubMed2006

Induction of neutrophil extracellular DNA lattices by placental microparticles and IL-8 and their presence in preeclampsia.

Preeclampsia, a severe pregnancy-related disorder, involves an overt activation of the maternal innate immune system, proposed to result from the elevated release of inflammatory syncytiotrophoblast microparticles (STBM) and cytokines from underlying placental anomaly involving abnormal trophoblast differentiation. Activation of circulating neutrophils has recently been shown to lead to the generation of fibrous extracellular lattices containing DNA, termed NETs (neutrophil extracellular traps). Therefore, we examined whether placentally derived factors activated peripheral neutrophils to generate NETs, and whether NETs formation was increased in preeclamptic placenta. Activation of isolated circulatory neutrophils on treatment with placentally derived factors (interleukin-8 and STBM) was assessed by measuring CD11b expression using Flow cytometry. Furthermore, NETs generation by these activated neutrophils in vitro and in vivo in the placental tissue sections were examined using electron microscopy and fluorescence microscopy. We report that placentally-derived interleukin-8 and STBM efficiently activated neutrophils and triggered NETs formation. Large numbers of NETs were present directly in the intervillous space of preeclamptic placentae. NETs, therefore, appear to be an integral part of neutrophil activation, and their increased presence in preeclampsia suggests that NETs may play a role in the underlying pathology.

Cell-Derived Microparticles↗
PubMed2006

Genotyping fetal paternally inherited SNPs by MALDI-TOF MS using cell-free fetal DNA in maternal plasma: influence of size fractionation.

The determination of fetal point mutations from fetal cell-free DNA (cf-DNA) in maternal plasma is technically challenging due to the preponderance of maternal sequences. It has recently been shown that fetal cf-DNA sequences are smaller than maternal ones and that the selection of small cf-DNA fragments by size fractionation by agarose gel electrophoresis leads to the enrichment of fetal cf-DNA sequences, thereby permitting the detection of otherwise masked fetal point mutations. In a separate development, the use of MALDI-TOF MS has also been shown to facilitate the detection of fetal point mutations from cf-DNA in maternal plasma. In this study, a combination of these approaches was examined. cf-DNA was extracted from 18 maternal plasma samples, 10 taken at term and 8 obtained early in the second trimester. A total of 41 SNP loci were examined in size-fractionated and total cf-DNA using either a conventional homogeneous MassEXTEND (hME) assay or a nucleotide-specific single allele base extension reaction (SABER) assay. The analysis of total cf-DNA indicated that size fractionation considerably enhanced the sensitivity of the standard hME assay, especially for samples taken early in pregnancy. Size fractionation also rendered the signals obtained by the SABER assay more precise.

Alleles↗
PubMed2006

Direct quantification of fetal cells in maternal blood by real-time PCR.

BACKGROUND: Fetal cells in maternal blood still present an enticing alternative for the development of a safe and efficacious non-invasive method for prenatal diagnosis. However, most enrichment methods are very tedious and have failed to realise this long sought after goal. We developed a simple, robust TaqMan real-time PCR assay to directly quantify male fetal cells in maternal blood using the multi-copy DYS14, without the need for any additional enrichment procedure. METHODS: The sensitivity of the DYS14 assay was evaluated by using female genomic DNA spiked with male DNA or male cells. The specificity of the DYS14 assay was evaluated by examining 40 adult blood samples and 44 maternal blood samples from pregnant women with known fetal gender. Direct quantification of fetal cells in maternal blood was performed in 14 of the maternal blood samples by the DYS14 assay. The results were compared to those by SRY assay. RESULTS: The sensitivity of DYS14 PCR assay was found to be higher than that of SRY assay for the detection of fetal cells. The number of fetal cells in maternal blood did not exceed 2/mL blood. The presence of cell-free fetal DNA in maternal blood could lead to erroneous quantification of fetal cell DNA. The number of fetal cells detected in blood cell pellets, which had been unwashed to remove the cell-free fetal DNA, was indeed significantly higher than those in extensively washed samples. CONCLUSIONS: We quantified the fetal cells in maternal blood without fetal cell enrichment procedures by TaqMan real-time PCR for a multi-copy DYS14 locus. Our assay is sensitive and also suitable for automation, and may be a useful tool for the determination of fetal-maternal cell trafficking, such as microchimerism.

Chromosomes, Human, Y↗
PubMed2006

Comparison of activin A and cell-free fetal DNA levels in maternal plasma from patients at high risk for preeclampsia.

OBJECTIVES: We examined the concentration of activin A in a prospective manner before the clinical manifestation of preeclampsia and compared the data with those of cell-free fetal DNA in the maternal plasma. METHODS: The levels of activin A were analysed by enzyme-linked immunosorbent assay (ELISA) for pregnant women: (1) with preeclampsia (n = 34) in the third-trimester and normal controls (n = 44); and (2) at-risk of preeclampsia in the second-trimester (n = 15) as indicated by uterine artery Doppler and normal controls (n = 68). Correlation between activin A level and cell-free fetal DNA level was examined using the Spearman rank test. RESULTS: The level of plasma activin A was significantly higher in the preeclamptic samples (12.056 vs 7.068 ng/mL, p = 0.000). The increase in the activin A concentration was observed prior to the onset of preeclampsia (3.483 vs 1.324 ng/mL, p = 0.000). This increase in activin A correlated significantly with the increased level of cell-free fetal DNA, in the maternal circulation prior to the onset of preeclampsia (r = 0.977, p = 0.000). CONCLUSION: Our data suggest that circulatory activin A could be an independent biomarker for the early identification and monitoring of preeclampsia.

Activins↗
PubMed2006

Reliable detection of trisomy 21 using MALDI-TOF mass spectrometry.

PURPOSE: Current diagnostic methods for chromosomal abnormalities rely mainly on karyotyping and occasionally fluorescent in situ hybridization or quantitative polymerase chain reaction. We describe an alternative molecular method for the detection of trisomy 21 involving mass spectrometric analysis of single nucleotide polymorphisms. METHODS: In collaboration with Sequenom, Inc., 350 blinded amniotic fluid, amniocyte culture, chorionic villus, or amniotic fluid supernatant samples were analyzed for trisomy 21 using SNP analysis and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Peak ratios were calculated for heterozygous genotypes and compared to control values generated from known euploid samples. An analytical algorithm using standard deviations from control values was used to determine the probability of a sample being affected or unaffected. RESULTS: Seventy-three trisomy 21 samples from among the 350 blinded samples were correctly identified. There were no false-positive or false-negative results among the complete trisomy 21 samples. One sample exhibiting mosaicism for trisomy 21 was identified as being unaffected. CONCLUSIONS: MALDI-TOF mass spectrometry is a robust and reproducible method for the detection of trisomy 21. Its amenability to high-throughput analysis and high degree of multiplexing make it a potential future diagnostic tool for the detection of other aneuploidies as well.

Algorithms↗
PubMed2006

Examination of maternal plasma erythropoietin and activin A concentrations with regard to circulatory erythroblast levels in normal and preeclamptic pregnancies.

OBJECTIVES: Preeclampsia has been shown to be associated with an increased number of fetal and maternal erythroblasts in the maternal circulation, suggesting that preeclampsia involves increased leakage of fetal cells across the placental barrier, as well as increased erythropoiesis. We examined the relationship between circulatory erythroblast levels with maternal plasma concentrations of erythropoietin and activin A. METHODS: In a case-control study, we examined 15 pregnancies affected by preeclampsia and 10 matched controls. Erythroblasts were enriched from maternal blood samples by magnetic cell sorting, enumerated and correlated with corresponding plasma activin A and erythropoietin concentrations. RESULTS: The proportion of erythroblast was elevated in preeclampsia (0.8 vs. 0.1%, p = 0.023). Erythropoietin and activin A concentrations were significantly elevated in preeclampsia (100.4 vs. 44.5 pg/ml, p = 0.023, and 7.4 vs. 1.85 ng/ml, p = 0.029, respectively). Circulatory erythroblast numbers were found to correlate with plasma activin A concentrations (r = 0.76, p = 0.01) in cases with preeclampsia. No such relationship existed for erythropoietin. CONCLUSIONS: Our data suggest that increased concentrations of activin A promote enhanced levels of erythropoiesis in preeclampsia. As the placenta is one of the major sources of activin A in pregnancy, this increase in activin A-dependent erythropoiesis in preeclampsia may be a reflection of an underlying placental hypoxic condition.

Activins↗
PubMed2006

Minimal alteration in the ratio of circulatory fetal DNA to fetal corticotropin-releasing hormone mRNA level in preeclampsia.

OBJECTIVES: We have recently observed that fetal DNA and fetal corticotropin-releasing hormone (CRH) mRNA are associated with in vitro generated syncytiotrophoblast-derived microparticles, and that the ratio of fetal DNA to mRNA (CRH) varied according to whether the particles were derived by predominantly apoptotic, apo-necrotic or necrotic pathways. Hence, we examined whether these ratios varied in maternal plasma samples taken from normotensive and preeclamptic pregnancies in vivo. METHODS: Maternal plasma samples were collected from 18 cases with preeclampsia and 29 normotensive term controls. Circulatory fetal CRH mRNA and DNA levels were quantified by real-time PCR and RT-PCR. RESULTS: Circulatory fetal mRNA and fetal DNA levels were significantly elevated in the preeclampsia study group when compared to normotensive controls. Alterations in the fetal mRNA to DNA ratio between the study and control groups were minimal, even when stratified into early (<34 weeks of gestation) and late (>34 weeks of gestation) onset preeclampsia. CONCLUSIONS: Our data suggest that although circulatory fetal DNA and mRNA levels are significantly elevated in preeclampsia, the ratios in maternal plasma are not dramatically altered.

Corticotropin-Releasing Hormone↗
PubMed2006

Cell-free DNA in maternal plasma: is it all a question of size?

Fetal cell-free DNA (cf-DNA) represents only a small fraction of the total cf-DNA in maternal plasma. This feature has rendered it difficult to reliably distinguish fetal alleles which are not very disparate from maternal ones, such as those involving point mutations, by conventional polymerase chain reaction (PCR)-based approaches. It has recently been shown that cell-free fetal DNA molecules have a smaller size than comparable cf-DNA molecules of maternal origin, and that this feature can be exploited for the selective enrichment of fetal DNA sequences, thereby permitting the detection of otherwise masked fetal genetic traits. By the use of this approach, we have shown that it is possible to detect fetal genetic loci for microsatellite markers, as well as point mutations involved in disorders such as achondroplasia and beta-thalassemia.

Achondroplasia↗
PubMed2006

Occurrence of neutrophil extracellular DNA traps (NETs) in pre-eclampsia: a link with elevated levels of cell-free DNA?

Manifest pre-eclampsia is associated with activation of peripheral neutrophils as well as elevations in maternal cell-free DNA. For this reason, we were very intrigued by recent reports indicating that activated circulatory neutrophils secrete nuclear DNA to generate extracellular DNA lattices, termed NETs (neutrophil extracellular traps). Our preliminary data indicate that placental syncytiotrophoblast microparticles, which are released in elevated amounts in pre-eclampsia, can induce NETs in isolated neutrophils. Furthermore, we found evidence for the increased presence of NETs directly in the intervillous space of pre-eclamptic placentae. Therefore, these newly discovered entities may be implicated in the underlying etiology of this disorder.

DNA↗
PubMed2006

Detection of SNPs in the plasma of pregnant women and in the urine of kidney transplant recipients by mass spectrometry.

Recently, it has been discovered that cell-free fetal DNA is smaller than corresponding maternal DNA. Therefore, circulating fetal DNA can be enriched by size-fractionation. Such a selection improves the non-invasive prenatal diagnosis of paternally inherited single gene mutations. Recent studies showed that MALDI-TOF mass spectrometry (MS) can be used to reliably detect fetal-specific single-nucleotide polymorphisms (SNPs) in maternal plasma. In this study, we looked at whether the size-fractionation approach could improve the detection of paternally inherited SNPs by MS assay. Our results indicated that the size-fractionation approach improved the analysis of paternally inherited SNP alleles. Our previous studies showed that donor-derived STR sequences could be detected in the urine of kidney transplant recipients. Here, we also examined whether donor-specific SNPs could be detected in recipient's urine by MS.

DNA↗
PubMed2006

Improvement of methods for the isolation of cell-free fetal DNA from maternal plasma: comparison of a manual and an automated method.

The low amount of cell-free fetal DNA present in the maternal circulation poses significant challenges to its use in future diagnostic applications, and ways of increasing the yield of this potential marker extracted from maternal plasma are constantly being explored. In this study, we compared two methods of DNA extraction, a manual and an automated method. Our analysis revealed that although the manual method yielded overall more total cell-free DNA, the automated system yielded higher quantities of cell-free DNA of fetal origin. Furthermore, the DNA isolated using the automated system appeared to be of greater purity than that isolated by the manual method, with fewer inhibitors to downstream real-time PCR reactions.

DNA↗
PubMed2006

Optimized real-time quantitative PCR measurement of male fetal DNA in maternal plasma.

DNA of fetal origin is present in the plasma of pregnant women. The quantitative measurement of circulatory fetal DNA (cfDNA) by real-time quantitative PCR (qPCR) has been applied to investigate a possible correlation between increased levels and pregnancy-related disorders. However, as the levels of cfDNA are close to the detection limit (LOD) of the method used, the measurements may not be reliable. This is also problematic for the evaluation of preanalytical steps, such as DNA extraction and cfDNA enrichment by size separation. We optimized a protocol for the qPCR analysis of the multi-copy sequence DYS14 on the Y chromosome. This was compared with an established assay for the single-copy SRY gene. Probit regression analysis showed that the limit of detection (LOD) of the DYS14 assay, (0.4 genome equivalents (GE)) and limit of quantification (LOQ) were 10-fold lower in comparison to SRY (4 GE). The levels of cfDNA obtained from the first trimester of pregnancy could be quantified with high precision by the DYS14 assay (CV below 25%) as opposed to the SRY measurements (26-140%). Additionally, fetal sex was correctly determined in all instances. The low copy numbers of fetal DNA in plasma of women in the first trimester of pregnancy can be measured reliably, targeting the DYS14 that is present in multiple copies per Y chromosome.

DNA↗