PubMed Health⌕ Search

Biomedical subjects

Rossa W K Chiu

Publications and source records attributed to Rossa W K Chiu.

At least 19 recordsLinked to original sources
PubMed2007

Plasma placental RNA allelic ratio permits noninvasive prenatal chromosomal aneuploidy detection.

Current methods for prenatal diagnosis of chromosomal aneuploidies involve the invasive sampling of fetal materials using procedures such as amniocentesis or chorionic villus sampling and constitute a finite risk to the fetus. Here, we outline a strategy for fetal chromosome dosage assessment that can be performed noninvasively through analysis of placental expressed mRNA in maternal plasma. We achieved noninvasive prenatal diagnosis of fetal trisomy 21 by determining the ratio between alleles of a single-nucleotide polymorphism (SNP) in PLAC4 mRNA, which is transcribed from chromosome 21 and expressed by the placenta, in maternal plasma. PLAC4 mRNA in maternal plasma was fetal derived and cleared after delivery. The allelic ratios in maternal plasma correlated with those in the placenta. Fetal trisomy 21 was detected noninvasively in 90% of cases and excluded in 96.5% of controls.

Asian People↗
PubMed2006

Prenatal diagnosis: progress through plasma nucleic acids.

Over the past 40 years, much effort has been spent on developing non-invasive prenatal diagnostic methods. Since 1997, the progress of this field has been accelerated by the unexpected finding of extracellular fetal nucleic acids in maternal plasma. These developments have been translated into many novel genetic, epigenetic and gene-expression markers, and are expected to have a fundamental impact on the future practice of prenatal diagnosis.

DNA↗
PubMed2006

Hypermethylated RASSF1A in maternal plasma: A universal fetal DNA marker that improves the reliability of noninvasive prenatal diagnosis.

BACKGROUND: We recently demonstrated that the promoter of the RASSF1A gene is hypermethylated in the placenta and hypomethylated in maternal blood cells. This methylation pattern allows the use of methylation-sensitive restriction enzyme digestion for detecting the placental-derived hypermethylated RASSF1A sequences in maternal plasma. METHODS: We performed real-time PCR after methylation-sensitive restriction enzyme digestion to detect placental-derived RASSF1A sequences in the plasma of 28 1st-trimester and 43 3rd-trimester pregnant women. We used maternal plasma to perform prenatal fetal rhesus D (RhD) blood group typing for 54 early-gestation RhD-negative women, with hypermethylated RASSF1A as the positive control for fetal DNA detection. RESULTS: Hypermethylated RASSF1A sequences were detectable in the plasma of all 71 pregnant women. The genotype of plasma RASSF1A after enzyme digestion was identical to the fetal genotype in each case, thus confirming its fetal origin. Nineteen of the 54 pregnant women undergoing prenatal fetal RhD genotyping showed undetectable RHD sequences in their plasma DNA samples. The fetal DNA control, RASSF1A, was not detectable in 4 of the 19 women. Subsequent chorionic villus sample analysis revealed that 2 of these 4 women with negative RHD and RASSF1A signals were in fact carrying RhD-positive fetuses. CONCLUSIONS: Hypermethylated RASSF1A is a universal marker for fetal DNA and is readily detectable in maternal plasma. When applied to prenatal RhD genotyping, this marker allows the detection of false-negative results caused by low fetal DNA concentrations in maternal plasma. This new marker can also be applied to many other prenatal diagnostic and monitoring scenarios.

Actins↗
PubMed2006

Noninvasive prenatal detection of fetal trisomy 18 by epigenetic allelic ratio analysis in maternal plasma: Theoretical and empirical considerations.

BACKGROUND: The discovery of cell-free fetal DNA in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis. However, the use of maternal plasma fetal DNA for the direct detection of fetal chromosomal aneuploidies has not been reported. We postulate that the aneuploidy status of a fetus could be revealed by an epigenetic allelic ratio approach, i.e., by analyzing the allelic ratio of a single-base variation present within DNA molecules exhibiting a placental-specific epigenetic signature in maternal plasma. METHODS: Placental-derived fetal-specific unmethylated maspin (SERPINB5) promoter sequences on human chromosome 18 were detectable in placental-maternal DNA mixtures and in maternal plasma by bisulfite modification followed by methylation-specific PCR (MSP) and primer extension. The ratios between the extension products of the 2 alleles were calculated for heterozygous placentas, placental-maternal blood cell DNA mixtures, and maternal plasma samples. The allelic ratios were compared between pregnancies carrying trisomy 18 and euploid fetuses. RESULTS: The epigenetic allelic ratios of all tested trisomy 18 samples deviated from the reference range obtained from euploid samples (placental DNA, 1.135 to 2.052; placental-maternal DNA mixtures, 1.170 to 1.985; maternal plasma, 0.330 to 3.044; without skew correction on the raw mass spectrometric data). A theoretical model was established and validated that predicted that a minimum of 200 copies of genomic DNA after bisulfite conversion were required for distinguishing euploid and aneuploid fetuses with confidence. CONCLUSION: Epigenetic allelic ratio analysis of maternal plasma DNA represents a promising approach for noninvasive prenatal diagnosis of fetal chromosomal aneuploidies.

Chromosomes, Human, Pair 18↗
PubMed2006

Plasma beta-globin DNA as a prognostic marker in chest pain patients.

BACKGROUND: Acute coronary syndrome may involve cell death and the release of nucleic acids into the circulation. We thus investigated whether plasma DNA concentrations are increased and determined its prognostic significance in patients with ACS. METHODS: Real-time polymerase chain reaction was used to quantitatively measure the beta-globin gene from blood samples taken from patients presenting to an emergency department with chest pain of probable cardiac cause. RESULTS: Samples from 58 patients with chest pain, and from 21 age- and sex-matched healthy control subjects were analysed. Compared with the control group, median plasma DNA concentrations were increased 1.5-fold in patients with minor cardiac injury, were increased further in patients with STEA and STEMI, and were the highest in those patients who died within 2 years (P=0.0005; post-hoc Dunn's, P<0.05). Median plasma DNA concentrations were higher in patients who later developed heart failure (1060 vs. 500 kGE/l; P=0.0095); higher in patients who later reinfarcted (1000 vs. 530 kGE/l; P=0.0298); higher in patients who had a cardiac arrest in that admission (1350 vs. 525 kGE/l; P=0.04); and were higher in patients who were readmitted within 6 months of discharge (725 vs. 475 kGE/l; P=0.04). CONCLUSION: Plasma DNA is a potential marker for post-ACS complications.

Adult↗
PubMed2006

Automated extraction protocol for quantification of SARS-coronavirus RNA in serum: an evaluation study.

BACKGROUND: We have previously developed a test for the diagnosis and prognostic assessment of the severe acute respiratory syndrome (SARS) based on the detection of the SARS-coronavirus RNA in serum by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR). In this study, we evaluated the feasibility of automating the serum RNA extraction procedure in order to increase the throughput of the assay. METHODS: An automated nucleic acid extraction platform using the MagNA Pure LC instrument (Roche Diagnostics) was evaluated. We developed a modified protocol in compliance with the recommended biosafety guidelines from the World Health Organization based on the use of the MagNA Pure total nucleic acid large volume isolation kit for the extraction of SARS-coronavirus RNA. The modified protocol was compared with a column-based extraction kit (QIAamp viral RNA mini kit, Qiagen) for quantitative performance, analytical sensitivity and precision. RESULTS: The newly developed automated protocol was shown to be free from carry-over contamination and have comparable performance with other standard protocols and kits designed for the MagNA Pure LC instrument. However, the automated method was found to be less sensitive, less precise and led to consistently lower serum SARS-coronavirus concentrations when compared with the column-based extraction method. CONCLUSION: As the diagnostic efficiency and prognostic value of the serum SARS-CoV RNA RT-PCR test is critically associated with the analytical sensitivity and quantitative performance contributed both by the RNA extraction and RT-PCR components of the test, we recommend the use of the column-based manual RNA extraction method.

Humans↗
PubMed2006

Serum proteomic fingerprints of adult patients with severe acute respiratory syndrome.

BACKGROUND: Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a new coronavirus strain, SARS-CoV. Specific proteomic patterns might be present in serum in response to the infection and could be useful for early detection of the disease. METHODS: Using surface-enhanced laser desorption/ionization (SELDI) ProteinChip technology, we profiled and compared serum proteins of 39 patients with early-stage SARS infection and 39 non-SARS patients who were suspected cases during the SARS outbreak period. Proteomic patterns associated with SARS were identified by bioinformatic and biostatistical analyses. Features of interest were then purified and identified by tandem mass spectrometry. RESULTS: Twenty proteomic features were significantly different between the 2 groups. Fifteen were increased in the SARS group, and 5 were decreased. Their concentrations were correlated with 2 or more clinical and/or biochemical variables. Two were correlated with the SARS-CoV viral load. Hierarchical clustering analysis showed that a majority of the SARS patients (95%) had similar serum proteomic profiles and identified 2 subgroups with poor prognosis. ROC curve analysis identified individual features as potential biomarkers for SARS diagnosis (areas under ROC curves, 0.733-0.995). ROC curve areas were largest for an N-terminal fragment of complement C3c alpha chain (m/z 28 119) and an internal fragment of fibrinogen alpha-E chain (m/z 5908). Immunoglobulin kappa light chain (m/z 24 505) positively correlated with viral load. CONCLUSIONS: Specific proteomic fingerprints in the sera of adult SARS patients could be used to identify SARS cases early during onset with high specificity and sensitivity.

Adult↗
PubMed2006

Time profile of appearance and disappearance of circulating placenta-derived mRNA in maternal plasma.

BACKGROUND: Fetal RNA of placental origin has been detected in the plasma of pregnant women, but the timing of the first appearance and the detailed kinetics of postdelivery clearance of such circulating RNA have not been studied. METHODS: To address the timing of the first appearance of circulating placental RNA, we collected serial maternal blood samples from 47 women who had conceived by assisted reproductive procedures. To address the postdelivery clearance kinetics, we collected serial postdelivery blood samples from 6 pregnant women who had delivered by cesarean section. Placenta-derived transcripts were sought by real-time quantitative reverse transcription-PCR. RESULTS: The earliest gestational age at which human placental lactogen and human chorionic gonadotropin beta-subunit mRNAs were detectable in a proportion of the pregnant women was the 4th week of gestation. The postdelivery study indicated that the median apparent half-life for the clearance of human placental lactogen mRNA was 14 min. CONCLUSIONS: Placenta-derived mRNA can be found in maternal plasma from very early on in gestation, suggesting a possible role for early noninvasive prenatal diagnosis or monitoring. The rapid kinetics of circulating placental mRNA suggest that its plasma concentrations may be used to monitor recent physiologic or pathologic events.

Female↗
PubMed2006

Noninvasive prenatal diagnosis by analysis of fetal DNA in maternal plasma.

Prenatal diagnosis has become a standard part of obstetrics care. Genetic diagnoses are established prenatally through the sampling of fetal genetic material by invasive methods such as amniocentesis or chorionic villus sampling, which are associated with a risk of fetal loss. Hence, the recent discovery of the presence of fetal DNA in maternal plasma has led to exciting possibilities of noninvasive prenatal diagnosis. Numerous applications have since been reported for the analysis of circulating fetal DNA. The accuracy of fetal DNA assessment from maternal plasma is dependent on the appropriate preanalytical handling of maternal blood samples, an efficient fetal DNA extraction protocol, and a sensitive and specific detection method. The protocol that has been applied regularly in the laboratory of the authors for the reliable detection and quantification of circulating fetal DNA is presented in this chapter.

Calibration↗
PubMed2006

Genomic sequencing of the severe acute respiratory syndrome-coronavirus.

The polymerase chain reaction (PCR), which can exponentially replicate a target DNA sequence, has formed the basis for the sensitive and direct examination of clinical samples for evidence of infection. During the epidemic of severe acute respiratory syndrome (SARS) in 2003, PCR not only offered a rapid way to diagnose SARS-coronavirus (SARS-CoV) infection, but also made the molecular analysis of its genomic sequence possible. Sequence variations were observed in the SAR-CoV obtained from different patients in this epidemic. These unique viral genetic signatures can be applied as a powerful molecular tool in tracing the route of transmission and in studying the genome evolution of SARS-CoV. To extract this wealth of information from the limited primary clinical specimens of SARS patients, we were presented with the challenge of efficiently amplifying fragments of the SARS-CoV genome for analysis. In this chapter, we will discuss how we managed to accomplish this task with our optimized protocols on reverse-transcription, nested PCR amplification, and DNA cycle sequencing. We will also discuss the sequence variations that typified some strains of SARS-CoV in the different phases during this epidemic. PCR amplification of the viral sequence and genomic sequencing of these critical sequence variations of re-emerging SARS-CoV strains would give us quick insights into the virus.

DNA Primers↗
PubMed2005

A simple and rapid approach for screening of SARS-coronavirus genotypes: an evaluation study.

BACKGROUND: The Severe Acute Respiratory Syndrome (SARS) was a newly emerged infectious disease which caused a global epidemic in 2002-2003. Sequence analysis of SARS-coronavirus isolates revealed that specific genotypes predominated at different periods of the epidemic. This information can be used as a footprint for tracing the epidemiology of infections and monitor viral evolution. However, direct sequencing analysis of a large number of clinical samples is cumbersome and time consuming. We present here a simple and rapid assay for the screening of SARS-coronavirus genotypes based on the use of fluorogenic oligonucleotide probes for allelic discrimination. METHODS: Thirty SARS patients were recruited. Allelic discrimination assays were developed based on the use of fluorogenic oligonucleotide probes (TaqMan). Genotyping of the SARS-coronavirus isolates obtained from these patients were carried out by the allelic discrimination assays and confirmed by direct sequencing. RESULTS: Genotyping based on the allelic discrimination assays were fully concordant with direct sequencing. All of the 30 SARS-coronavirus genotypes studied were characteristic of genotypes previously documented to be associated with the latter part of the epidemic. Seven of the isolates contained a previously reported major deletion but in patients not epidemiologically related to the previously studied cohort. CONCLUSION: We have developed a simple and accurate method for the characterization and screening of SARS-coronavirus genotypes. It is a promising tool for the study of epidemiological relationships between documented cases during an outbreak.

Alleles↗
PubMed2005

Detection of the placental epigenetic signature of the maspin gene in maternal plasma.

The discovery of fetal DNA in the plasma of pregnant women has opened up new approaches for noninvasive prenatal diagnosis and monitoring. Up to now, the lack of a fetal DNA marker that can be universally detected in maternal plasma has limited the clinical application of this technology. We hypothesized that epigenetic differences between the placenta and maternal blood cells could be used for developing such a marker. By using bisulfite DNA sequencing, the methylation status of the maspin gene promoter in placental tissues and paired maternal blood cells from pregnant women was analyzed. The maspin gene promoter was found to be hypomethylated in placental tissues and densely methylated in maternal blood cells. Genotyping of a single nucleotide polymorphism within the unmethylated maspin sequences in maternal plasma demonstrated that these sequences were derived from the fetus. By using real-time quantitative methylation-specific PCR, unmethylated maspin sequences were detected in maternal plasma in all three trimesters of pregnancy and were cleared within 24 h after delivery. The maternal plasma concentration of unmethylated maspin sequences was elevated by a median of 5.7 times in preeclamptic pregnancies compared with nonpreeclamptic pregnancies. Hypomethylated maspin DNA is the first universal marker for fetal DNA in maternal plasma, thus allowing the measurement of fetal DNA concentrations in pregnancy-associated disorders, irrespective of fetal gender and genetic polymorphisms. Differential DNA methylation between the placenta and maternal blood cells may be exploited to develop further markers for noninvasive prenatal assessment.

Adult↗
PubMed2005

Circulating placental RNA in maternal plasma is associated with a preponderance of 5' mRNA fragments: implications for noninvasive prenatal diagnosis and monitoring.

BACKGROUND: The molecular characteristics of placental RNA circulating in maternal plasma are unknown. We investigated the integrity of circulating placental RNA in maternal plasma and tested the relevance of plasma RNA integrity for noninvasive prenatal diagnosis. METHODS: Six different placental transcripts and mRNA of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantified for the 5' and 3' regions in maternal plasma by 1-step real-time reverse transcription-PCR (RT-PCR) assays. This quantitative strategy was validated by 2-step RT-PCR and serial dilution experiments. The rates of detection by the 5' and 3' assays for the beta-subunit of human chorionic gonadotropin (beta hCG) were assessed in maternal plasma samples collected from different gestational periods. RESULTS: For 5 of the 7 genes, the plasma mRNA concentrations measured by the 5' amplicons were significantly higher than those measured by the corresponding 3' amplicons. Every transcript under study demonstrated a higher rate of detection in the 5' assay than in the 3' assay in maternal plasma. In particular, the detection rate of beta hCG mRNA in maternal plasma was increased throughout gestation when the 5' assay was used. CONCLUSIONS: Circulating placental RNA is associated with a preponderance of 5' mRNA fragments in maternal plasma. Apart from its intrinsic biological interest, this information could have important implications for the development of new assays targeting fetal RNA markers for noninvasive prenatal diagnosis and monitoring.

Chorionic Gonadotropin↗
PubMed2005

Absence of association between angiotensin converting enzyme polymorphism and development of adult respiratory distress syndrome in patients with severe acute respiratory syndrome: a case control study.

BACKGROUND: It has been postulated that genetic predisposition may influence the susceptibility to SARS-coronavirus infection and disease outcomes. A recent study has suggested that the deletion allele (D allele) of the angiotensin converting enzyme (ACE) gene is associated with hypoxemia in SARS patients. Moreover, the ACE D allele has been shown to be more prevalent in patients suffering from adult respiratory distress syndrome (ARDS) in a previous study. Thus, we have investigated the association between ACE insertion/deletion (I/D) polymorphism and the progression to ARDS or requirement of intensive care in SARS patients. METHOD: One hundred and forty genetically unrelated Chinese SARS patients and 326 healthy volunteers were recruited. The ACE I/D genotypes were determined by polymerase chain reaction and agarose gel electrophoresis. RESULTS: There is no significant difference in the genotypic distributions and the allelic frequencies of the ACE I/D polymorphism between the SARS patients and the healthy control subjects. Moreover, there is also no evidence that ACE I/D polymorphism is associated with the progression to ARDS or the requirement of intensive care in the SARS patients. In multivariate logistic analysis, age is the only factor associated with the development of ARDS while age and male sex are independent factors associated with the requirement of intensive care. CONCLUSION: The ACE I/D polymorphism is not directly related to increased susceptibility to SARS-coronavirus infection and is not associated with poor outcomes after SARS-coronavirus infection.

Adult↗
PubMed2005

Clinical, virologic and immunologic profiles of a young infant with severe acute respiratory syndrome.

The clinical findings, plasma viral load, cytokines and chemokines of a 4-month-old infant with severe acute respiratory syndrome (SARS) were assessed at different phases of the disease. Ribavirin failed to inhibit SARS coronavirus (SARS-CoV) replication. One-step real time reverse transcription-polymerase chain reaction for plasma SARS-CoV RNA quantification was useful for early diagnosis and monitoring viremia.

Cytokines↗