Tze K. Lau

Bio: Tze K. Lau is an academic researcher from The Chinese University of Hong Kong. The author has contributed to research in topics: Prenatal diagnosis & Trisomy. The author has an hindex of 44, co-authored 69 publications receiving 14779 citations.

Quantitative Analysis of Fetal DNA in Maternal Plasma and Serum: Implications for Noninvasive Prenatal Diagnosis

Abstract: Summary We have developed a real-time quantitative PCR assay to measure the concentration of fetal DNA in maternal plasma and serum. Our results show that fetal DNA is present in high concentrations in maternal plasma, reaching a mean of 25.4 genome equivalents/ml (range 3.3–69.4) in early pregnancy and 292.2 genome equivalents/ml (range 76.9–769) in late pregnancy. These concentrations correspond to 3.4% (range 0.39%–11.9%) and 6.2% (range 2.33%–11.4%) of the total plasma DNA in early and late pregnancy, respectively. Sequential follow-up study of women who conceived by in vitro fertilization shows that fetal DNA can be detected in maternal serum as early as the 7th wk of gestation and that it then increases in concentration as pregnancy progresses. These data suggest that fetal DNA can be readily detected in maternal plasma and serum and may be a valuable source of material for noninvasive prenatal diagnosis.

Rapid Clearance of Fetal DNA from Maternal Plasma

Abstract: Summary Fetal DNA has been detected in maternal plasma during pregnancy. We investigated the clearance of circulating fetal DNA after delivery, using quantitative PCR analysis of the sex-determining region Y gene as a marker for male fetuses. We analyzed plasma samples from 12 women 1–42 d after delivery of male babies and found that circulating fetal DNA was undetectable by day 1 after delivery. To obtain a higher time-resolution picture of fetal DNA clearance, we performed serial sampling of eight women, which indicated that most women (seven) had undetectable levels of circulating fetal DNA by 2 h postpartum. The mean half-life for circulating fetal DNA was 16.3 min (range 4–30 min). Plasma nucleases were found to account for only part of the clearance of plasma fetal DNA. The rapid turnover of circulating DNA suggests that plasma DNA analysis may be less susceptible to false-positive results, which result from carryover from previous pregnancies, than is the detection of fetal cells in maternal blood; also, rapid turnover may be useful for the monitoring of feto-maternal events with rapid dynamics. These results also may have implications for the study of other types of nonhost DNA in plasma, such as circulating tumor-derived and graft-derived DNA in oncology and transplant patients, respectively.

Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma.

Abstract: Chromosomal aneuploidy is the major reason why couples opt for prenatal diagnosis. Current methods for definitive diagnosis rely on invasive procedures, such as chorionic villus sampling and amniocentesis, and are associated with a risk of fetal miscarriage. Fetal DNA has been found in maternal plasma but exists as a minor fraction among a high background of maternal DNA. Hence, quantitative perturbations caused by an aneuploid chromosome in the fetal genome to the overall representation of sequences from that chromosome in maternal plasma would be small. Even with highly precise single molecule counting methods such as digital PCR, a large number of DNA molecules and hence maternal plasma volume would need to be analyzed to achieve the necessary analytical precision. Here we reasoned that instead of using approaches that target specific gene loci, the use of a locus-independent method would greatly increase the number of target molecules from the aneuploid chromosome that could be analyzed within the same fixed volume of plasma. Hence, we used massively parallel genomic sequencing to quantify maternal plasma DNA sequences for the noninvasive prenatal detection of fetal trisomy 21. Twenty-eight first and second trimester maternal plasma samples were tested. All 14 trisomy 21 fetuses and 14 euploid fetuses were correctly identified. Massively parallel plasma DNA sequencing represents a new approach that is potentially applicable to all pregnancies for the noninvasive prenatal diagnosis of fetal chromosomal aneuploidies.

Maternal plasma DNA sequencing reveals the genome-wide genetic and mutational profile of the fetus

Abstract: Cell-free fetal DNA is present in the plasma of pregnant women. It consists of short DNA fragments among primarily maternally derived DNA fragments. We sequenced a maternal plasma DNA sample at up to 65-fold genomic coverage. We showed that the entire fetal and maternal genomes were represented in maternal plasma at a constant relative proportion. Plasma DNA molecules showed a predictable fragmentation pattern reminiscent of nuclease-cleaved nucleosomes, with the fetal DNA showing a reduction in a 166-base pair (bp) peak relative to a 143-bp peak, when compared with maternal DNA. We constructed a genome-wide genetic map and determined the mutational status of the fetus from the maternal plasma DNA sequences and from information about the paternal genotype and maternal haplotype. Our study suggests the feasibility of using genome-wide scanning to diagnose fetal genetic disorders prenatally in a noninvasive way.

Non-invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: Large scale validity study

Abstract: Objectives To validate the clinical efficacy and practical feasibility of massively parallel maternal plasma DNA sequencing to screen for fetal trisomy 21 among high risk pregnancies clinically indicated for amniocentesis or chorionic villus sampling. Design Diagnostic accuracy validated against full karyotyping, using prospectively collected or archived maternal plasma samples. Setting Prenatal diagnostic units in Hong Kong, United Kingdom, and the Netherlands. Participants 753 pregnant women at high risk for fetal trisomy 21 who underwent definitive diagnosis by full karyotyping, of whom 86 had a fetus with trisomy 21. Intervention Multiplexed massively parallel sequencing of DNA molecules in maternal plasma according to two protocols with different levels of sample throughput: 2-plex and 8-plex sequencing. Main outcome measures Proportion of DNA molecules that originated from chromosome 21. A trisomy 21 fetus was diagnosed when the z score for the proportion of chromosome 21 DNA molecules was >3. Diagnostic sensitivity, specificity, positive predictive value, and negative predictive value were calculated for trisomy 21 detection. Results Results were available from 753 pregnancies with the 8-plex sequencing protocol and from 314 pregnancies with the 2-plex protocol. The performance of the 2-plex protocol was superior to that of the 8-plex protocol. With the 2-plex protocol, trisomy 21 fetuses were detected at 100% sensitivity and 97.9% specificity, which resulted in a positive predictive value of 96.6% and negative predictive value of 100%. The 8-plex protocol detected 79.1% of the trisomy 21 fetuses and 98.9% specificity, giving a positive predictive value of 91.9% and negative predictive value of 96.9%. Conclusion Multiplexed maternal plasma DNA sequencing analysis could be used to rule out fetal trisomy 21 among high risk pregnancies. If referrals for amniocentesis or chorionic villus sampling were based on the sequencing test results, about 98% of the invasive diagnostic procedures could be avoided.

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

Abstract: 抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number

Abstract: Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ∼2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100 000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associat...

DNA Fragments in the Blood Plasma of Cancer Patients: Quantitations and Evidence for Their Origin from Apoptotic and Necrotic Cells

Abstract: Increased levels of DNA fragments have frequently been found in the blood plasma of cancer patients. Published data suggest that only a fraction of the DNA in blood plasma is derived from cancer cells. However, it is not known how much of the circulating DNA is from cancer or from noncancer cells. By quantitative methylation-specific PCR of the promoter region of the CDKN2A tumor suppressor gene, we were able to quantify the fraction of plasma DNA derived from tumor cells. In the plasma samples of 30 unselected cancer patients, we detected quantities of tumor DNA from only 3% to as much as 93% of total circulating DNA. We investigated possible origins of nontumor DNA in the plasma and demonstrate here a contribution of T-cell DNA in a few cases only. To investigate the possibility that plasma DNA originates from apoptotic or necrotic cells, we performed studies with apoptotic (staurosporine) and necrotic (staurosporine plus oligomycin) cells in vitro and with mice after induction of apoptotic (anti-CD95) or necrotic (acetaminophen) liver injury. Increasing amounts of DNA were found to be released in the supernatants of cells and in the blood plasma samples of treated animals. A clear discrimination of apoptotic and necrotic plasma DNA was possible by gel electrophoresis. The same characteristic patterns of DNA fragments could be identified in plasma derived from different cancer patients. The data are consistent with the possibility that apoptotic and necrotic cells are a major source for plasma DNA in cancer patients.

RNA sequencing: advances, challenges and opportunities

Abstract: In the few years since its initial application, massively parallel cDNA sequencing, or RNA-seq, has allowed many advances in the characterization and quantification of transcriptomes. Recently, several developments in RNA-seq methods have provided an even more complete characterization of RNA transcripts. These developments include improvements in transcription start site mapping, strand-specific measurements, gene fusion detection, small RNA characterization and detection of alternative splicing events. Ongoing developments promise further advances in the application of RNA-seq, particularly direct RNA sequencing and approaches that allow RNA quantification from very small amounts of cellular materials.

Analysis of Circulating Tumor DNA to Monitor Metastatic Breast Cancer

Abstract: Background The management of metastatic breast cancer requires monitoring of the tumor burden to determine the response to treatment, and improved biomarkers are needed. Biomarkers such as cancer antigen 15-3 (CA 15-3) and circulating tumor cells have been widely studied. However, circulating cell-free DNA carrying tumor-specific alterations (circulating tumor DNA) has not been extensively investigated or compared with other circulating biomarkers in breast cancer. Methods We compared the radiographic imaging of tumors with the assay of circulating tumor DNA, CA 15-3, and circulating tumor cells in 30 women with metastatic breast cancer who were receiving systemic therapy. We used targeted or whole-genome sequencing to identify somatic genomic alterations and designed personalized assays to quantify circulating tumor DNA in serially collected plasma specimens. CA 15-3 levels and numbers of circulating tumor cells were measured at identical time points. Results Circulating tumor DNA was successfully detected in 29 of the 30 women (97%) in whom somatic genomic alterations were identified; CA 15-3 and circulating tumor cells were detected in 21 of 27 women (78%) and 26 of 30 women (87%), respectively. Circulating tumor DNA levels showed a greater dynamic range, and greater correlation with changes in tumor burden, than did CA 15-3 or circulating tumor cells. Among the measures tested, circulating tumor DNA provided the earliest measure of treatment response in 10 of 19 women (53%). Conclusions This proof-of-concept analysis showed that circulating tumor DNA is an informative, inherently specific, and highly sensitive biomarker of metastatic breast cancer. (Funded by Cancer Research UK and others.)