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Yvonne Y-Y Or, Grace T-Y Chung,
Ka-Fai To,
Chit Chow,
Kwong-Wai Choy,
Carol Y-K Tong,
Alice W-C Leung,
Angela B-Y Hui,
Sai-Wah Tsao,
Ho-Keung Ng,
Timothy T-C Yip,
Pierre Busson,
Kwok-Wai Lo
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ABSTRACT: Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer commonly occurring in southern China. To decipher the molecular basis of this cancer, we performed high-resolution array CGH analysis on eight tumour lines and 10 primary tumours to identify the genes involved in NPC tumorigenesis. In this study, multiple regions of gain were consistently found at 1q21-q24, 7q11-12, 7q21-22., 11q13, 12p13, 12q13, 19p13 and 19q13. Importantly, a 2.1 Mb region at 12p13.31 was highly amplified in a NPC xenograft, xeno-2117. By FISH mapping, we have further delineated the amplicon to a 1.24 region flanked by RP11-319E16 and RP11-433J6. Copy number gains of this amplicon were confirmed in 21/41 (51%) primary tumours, while three cases (7.3%) showed high copy number amplification. Among the 13 genes within this amplicon, three candidate genes, lymphotoxin beta receptor (LTbetaR), tumour necrosis factor receptor superfamily memeber 1A (TNFRSF1R) and FLJ10665, were specifically over-expressed in the NPC xenograft with 12p13.3 amplification. However, only LTbetaR was frequently over-expressed in primary tumours. LTbetaR is a member of the TNF family of receptors, which can modulate NF-kappaB signalling pathways. Over-expression of LTbetaR in nasopharyngeal epithelial cells resulted in an increase of NF-kappaB activity and cell proliferation. In vivo study showed that suppression of LTbetaR by siRNA led to growth inhibition in the NPC tumour with 12p13.3 amplification. These findings implied that LTbetaR is a potential NPC-associated oncogene within the 12p13.3 amplicon and that its alteration is important in NPC tumorigenesis.
The Journal of Pathology 08/2009; 220(1):97-107. · 6.32 Impact Factor
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K C Allen Chan,
Chunming Ding,
Ageliki Gerovassili,
Sze W Yeung,
Rossa W K Chiu,
Tse N Leung,
Tze K Lau,
Stephen S C Chim, Grace T Y Chung,
Kypros H Nicolaides,
Y M Dennis Lo
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ABSTRACT: 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.
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.
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.
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.
Clinical Chemistry 01/2007; 52(12):2211-8. · 7.91 Impact Factor
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ABSTRACT: 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.
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.
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.
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.
BMC Infectious Diseases 02/2006; 6:20. · 3.12 Impact Factor
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Clinical Chemistry 07/2005; 51(6):1074-6. · 7.91 Impact Factor
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Clinical Chemistry 04/2005; 51(3):655-8. · 7.91 Impact Factor
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ABSTRACT: 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.
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.
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.
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.
BMC Infectious Diseases 02/2005; 5:87. · 3.12 Impact Factor
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K C Allen Chan,
Nelson L S Tang,
David S C Hui, Grace T Y Chung,
Alan K L Wu,
Stephen S C Chim,
Rossa W K Chiu,
Nelson Lee,
K W Choi,
Y M Sung,
Paul K S Chan,
Y K Tong,
S T Lai,
W C Yu,
Owen Tsang,
Y M Dennis Lo
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ABSTRACT: 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.
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.
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.
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.
BMC Infectious Diseases 02/2005; 5:26. · 3.12 Impact Factor
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JAMA The Journal of the American Medical Association 01/2005; 292(23):2835; author reply 2835-6. · 30.03 Impact Factor
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Rossa W K Chiu,
Nelson L S Tang,
David S C Hui, Grace T Y Chung,
Stephen S C Chim,
K C Allen Chan,
Ying-man Sung,
Louis Y S Chan,
Yu-kwan Tong,
Wing-shan Lee,
Paul K S Chan,
Y M Dennis Lo
Clinical Chemistry 10/2004; 50(9):1683-6. · 7.91 Impact Factor
