Yu Kwan Tong

Systematic micro-array based identification of placental mRNA in maternal plasma: Towards non-invasive prenatal gene expression profiling

The discovery of fetal DNA in the plasma of pregnant women1 has led to the development of promising approaches for non-invasive prenatal diagnosis.2–6 However, as fetal and maternal DNA species co-exist in maternal plasma, these DNA based diagnostic applications depend largely on the use of genetic markers that would allow the discrimination between fetal and maternal DNA (for example, the Y chromosome of a male fetus), and thus, a particular genetic marker could generally only be used in a proportion of pregnancies. This situation has prompted a quest by many laboratories to develop fetal nucleic acid markers that are independent of sex or polymorphism. The detection of fetal RNA in maternal plasma7 offers new possibilities for non-invasive prenatal investigation. This field has recently taken on new momentum as robust methods for plasma RNA extraction have been developed8 and circulating RNA has been shown to be surprisingly stable,9 possibly through an association with particulate matter.8 Furthermore, recent studies have identified the placenta as a significant source of such circulating fetal RNA.10 Hence, placental expressed mRNA transcripts, such as those coding for human placental lactogen ( hPL ), human chorionic gonadotropin β subunit ( βhCG ),10 and corticotropin releasing hormone ( CRH ),11 have been shown to be detectable in maternal plasma. Quantitative assays have been developed for the measurement of these circulating mRNA transcripts.8 The pregnancy specificity of these mRNA species has been demonstrated by their rapid clearance from maternal plasma after delivery.10,11 Thus, the detection in maternal plasma of mRNA transcripts derived from the plasma offers new avenues for the development of fetal specific nucleic acid markers that are independent of sex and polymorphism for the non-invasive prenatal assessment of all pregnancies.12 The clinical value of such an approach has …

Serum Proteomic Fingerprints of Adult Patients with Severe Acute Respiratory Syndrome

Abstract 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 α chain (m/z 28 119) and an internal fragment of fibrinogen α-E chain (m/z 5908). Immunoglobulin κ 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.

Genomic characterisation of the severe acute respiratory syndrome coronavirus of Amoy Gardens outbreak in Hong Kong

Summary Severe acute respiratory syndrome (SARS) is a global health concern. In Hong Kong, two major outbreaks, one hospital based and the other in the Amoy Gardens apartments, were identified. The frequency of diarrhoea, admission to intensive care, and mortality differed significantly between the two outbreaks. We did genomic sequencing for viral isolates from five Amoy Gardens patients. The virus sequence was identical in four of these five patients. The sequence data from one hospital case and the four identical community cases had only three nucleotide differences. Alterations in the SARS coronavirus genome are unlikely to have caused the distinctive clinical features of the Amoy Gardens patients, and these results highlight the importance of non-viral genomic factors in this outbreak.

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

BackgroundIt 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.MethodOne 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.ResultsThere 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.ConclusionThe 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.

ACE2 Gene Polymorphisms Do Not Affect Outcome of Severe Acute Respiratory Syndrome

Severe acute respiratory syndrome (SARS) is the first pandemic of the 21st century (1). Since its recognition, 8437 individuals have been affected and 813 have died (2). Approximately 20–30% of patients required intensive care admission (1). Although there was a slight predominance of female SARS patients, possibly because of the overrepresentation of female healthcare workers (1), male SARS patients were more likely to suffer poor outcomes (3). In a major hospital outbreak in Hong Kong (4), 32% of male and 15% of female SARS patients required intensive care or died. Remarkably, similar demographic data were seen among SARS patients in the greater Toronto area, Canada, where 32% of males and 14% of females with SARS required intensive care or died (5). Karlberg et al. (3) studied the case fatality rates among all confirmed SARS patients documented in the Hong Kong SARS epidemic in 2003. The authors concluded that the mortality rates differed significantly between males and females, being 21.9% and 13.2%, respectively. The relative risk for death in males was 1.62 after adjustment for age. It is thus an intriguing coincidence that ACE2 , the gene for the newly identified functional receptor for the SARS coronavirus, angiotensin-converting enzyme 2, maps to the X-chromosome (Xp22) (6). ACE2 was first identified as a homolog of angiotensin-converting enzyme with zinc metalloproteinase activity (7). Many of its activities differ from those of angiotensin-converting enzyme (8). ACE2 has been found …

Universal Haplotype-Based Noninvasive Prenatal Testing for Single Gene Diseases.

BACKGROUND Researchers have developed approaches for the noninvasive prenatal testing of single gene diseases. One approach that allows for the noninvasive assessment of both maternally and paternally inherited mutations involves the analysis of single nucleotide polymorphisms (SNPs) in maternal plasma DNA with reference to parental haplotype information. In the past, parental haplotypes were resolved by complex experimental methods or inferential approaches, such as through the analysis of DNA from other affected family members. Recently, microfluidics-based linked-read sequencing technology has become available and allows the direct haplotype phasing of the whole genome rapidly. We explored the feasibility of applying this direct haplotyping technology in noninvasive prenatal testing. METHODS We first resolved the haplotypes of parental genomes with the use of linked-read sequencing technology. Then, we identified SNPs within and flanking the genes of interest in maternal plasma DNA by targeted sequencing. Finally, we applied relative haplotype dosage analysis to deduce the mutation inheritance status of the fetus. RESULTS Haplotype phasing and relative haplotype dosage analysis of 12 out of 13 families were successfully achieved. The mutational status of these 12 fetuses was correctly classified. CONCLUSIONS High-throughput linked-read sequencing followed by maternal plasma-based relative haplotype dosage analysis represents a streamlined approach for noninvasive prenatal testing of inherited single gene diseases. The approach bypasses the need for mutation-specific assays and is not dependent on the availability of DNA from other affected family members. Thus, the approach is universally applicable to pregnancies at risk for the inheritance of a single gene disease.

Technical concerns about immunoprecipitation of methylated fetal DNA for noninvasive trisomy 21 diagnosis

Technical concerns about immunoprecipitation of methylated fetal DNA for noninvasive trisomy 21 diagnosis

Plasma epigenetic markers for cancer detection and prenatal diagnosis.

Successful detection of circulating nucleic acids has opened up new possibilities in cancer testing and prenatal diagnosis. Circulating DNA markers are useful in cancer detection, prognostication and monitoring. Cancer-associated molecular changes which can be detected include gene mutations, chromosomal rearrangements, microsatellite alterations, viral sequences, and, to be discussed in more detailed, gene promoter hypermethylation. Methylation analysis is commonly performed by DNA digestion with methylation-sensitive restriction endonucleases followed by polymerase chain reaction (PCR), or bisulfite modification followed by methylation-specific PCR (MSP). The detection of fetal DNA in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis. However, circulating fetal DNA detection has been based on exploiting gender and polymorphic differences between the fetus and mother. The recent discovery of epigenetic differences between the maternal and the fetal DNA detectable in maternal plasma has launched a hunt for fetal-derived epigenetic markers in maternal plasma. It is hoped that this type of universally applicable markers would be made available in a clinical diagnostic setting in the near future.

Serum Amyloid A Is Not Useful in the Diagnosis of Severe Acute Respiratory Syndrome

In our present study, we aimed to investigate whether the serum concentration of serum amyloid A (SAA), as measured by the surface-enhanced laser desorption/ionization (SELDI) ProteinChip technology or by ELISA, is useful in differentiating the patients with severe acute respiratory syndrome (SARS) from the non-SARS patients who were suspected cases during the SARS outbreak period. In a recent report from Kang et al. (1), the mean intensity of the protein peak at m/z 11514, identified by SELDI ProteinChip technology, of the SARS patient groups was 8 times greater than the intensity of the corresponding peak in the control patient group. This SELDI peak was observed previously in another proteomic study, under similar experimental conditions, and was identified as SAA (2). In a recent study by Yip et al.(3), the intensity of a SELDI peak at m/z 11695, which was identified as SAA, was significantly higher in the SARS patient group than in the control group. A similar increased peak was also found in pediatric patients with SARS (4). All of these studies suggested that SAA is useful in the diagnosis of SARS. In these studies, the control cases were either healthy persons or patients with viral …

Genomic Sequencing of a SARS Coronavirus Isolate That Predated the Metropole Hotel Case Cluster in Hong Kong

The epidemic of severe acute respiratory syndrome (SARS) swept across the globe, with reported cases in more than 30 countries. As of July 11, 2003, the number of reported probable cases was 8437, with 813 deaths (1). A novel coronavirus, SARS-CoV, was promptly implicated as the causative agent (2)(3)(4). Macaques infected with SARS-CoV subsequently developed respiratory symptoms and pathology similar to SARS patients, thus fulfilling the Koch postulates (5). Efforts in sequencing the viral genome promptly followed, and the genomic sequence revealed little homology to previously characterized strains of coronaviruses (6)(7). The complete genomic sequences of several SARS-CoV isolates have since been made publicly available (www.ncbi.nlm.nih.gov). Several sequence variations exist among isolates. In general, based on these sequence variations, the majority of the isolates can be segregated into two groups: isolates that were obtained from individuals who were epidemiologically linked to and those who were not linked to the Metropole Hotel in Hong Kong (8)(9)(10). Ruan et al. (10) compared the genomic sequences of 14 SARS-CoV isolates and suggested that a haplotype comprising four nucleotide positions, namely, 9404, 17564, 22222, and 27827 [GenBank accession no. AY274119 (7)], clearly defined two distinct genotypes. Isolates that were epidemiologically linked to the Metropole Hotel cluster have the configuration T:T:T:T, as opposed to the sequence C:G:C:C seen in the unassociated strains. (Note: The usage of the DNA-based code for the designation of SARS-CoV haplotypes does not imply that this virus possesses a DNA genome.) SARS was first reported in Guangdong Province, China, in November 2002 (11). Isolates that demonstrated the C:G:C:C haplotype were epidemiologically traceable to the early part of the epidemic (9). On the other hand, SARS …