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Rossa W K Chiu - One of the best experts on this subject based on the ideXlab platform.

  • fetal mitochondrial dna in Maternal Plasma in surrogate pregnancies detection and topology
    Prenatal Diagnosis, 2021
    Co-Authors: Maryjane L, K Allen C Chan, Peiyong Jiang, S A Yakovenko, Haiqiang Zhang, Suk Hang Cheng, V P Apryshko, Alex Zhavoronkov, Rossa W K Chiu
    Abstract:

    OBJECTIVES Due to the Maternally-inherited nature of mitochondrial DNA (mtDNA), there is a lack of information regarding fetal mtDNA in the Plasma of pregnant women. We aim to explore the presence and topologic forms of circulating fetal and Maternal mtDNA molecules in surrogate pregnancies. METHODS Genotypic differences between fetal and surrogate Maternal mtDNA were used to identify the fetal and Maternal mtDNA molecules in Plasma. Plasma samples were obtained from the surrogate pregnant mothers. Using cleavage-end signatures of BfaI restriction enzyme, linear and circular mtDNA molecules in Maternal Plasma could be differentiated. RESULTS Fetal-derived mtDNA molecules were mainly linear (median: 88%; range: 80%-96%), whereas approximately half of the Maternal-derived mtDNA molecules were circular (median: 51%; range: 42%-60%). The fetal DNA fraction of linear mtDNA was lower (median absolute difference: 9.8%; range: 1.1%-27%) than that of nuclear DNA (median: 20%; range: 9.7%-35%). The fetal-derived linear mtDNA molecules were shorter than the Maternal-derived ones. CONCLUSION Fetal mtDNA is present in Maternal Plasma, and consists mainly of linear molecules. Surrogate pregnancies represent a valuable clinical scenario for exploring the biology and potential clinical applications of circulating mtDNA, for example, for pregnancies conceived following mitochondrial replacement therapy.

  • noninvasive prenatal methylomic analysis by genomewide bisulfite sequencing of Maternal Plasma dna
    Clinical Chemistry, 2013
    Co-Authors: Fiona M F Lun, K Allen C Chan, Rossa W K Chiu, Tak Yeung Leung, Kun Sun, Peiyong Jiang, Hao Sun
    Abstract:

    BACKGROUND: Epigenetic mechanisms play an important role in prenatal development, but fetal tissues are not readily accessible. Fetal DNA molecules are present in Maternal Plasma and can be analyzed noninvasively. METHODS: We applied genomewide bisulfite sequencing via 2 approaches to analyze the methylation profile of Maternal Plasma DNA at single-nucleotide resolution. The first approach used Maternal blood samples and polymorphic differences between the mother and fetus to analyze the fetal methylome across the genome. The second approach used the methylation profile of Maternal blood cells and the fractional fetal DNA concentration in Maternal Plasma to deduce the placental methylomic profile from Maternal Plasma DNA-sequencing data. RESULTS: Because of the noninvasive nature of these approaches, we were able to serially assess the methylation profiles of fetal, placental, and Maternal Plasma with Maternal blood samples collected in the first and third trimesters and after delivery. Gestation-related changes were observed. The fetal methylation profile deduced from Maternal Plasma data resembled that of the placental methylome, both on a genomewide level and per CpG site. Imprinted genes and differentially methylated regions were identified from the Maternal Plasma data. We demonstrated one potential clinical application of Maternal Plasma bisulfite sequencing with the successful detection of fetal trisomy 21. CONCLUSIONS: We successfully analyzed fetal and placental methylomes on a genomewide scale, noninvasively and serially. This development offers a powerful method for research, biomarker discovery, and clinical testing for pregnancy-related disorders.

  • high resolution profiling of fetal dna clearance from Maternal Plasma by massively parallel sequencing
    Clinical Chemistry, 2013
    Co-Authors: Shara W Y Lee, K Allen C Chan, Tak Yeung Leung, Peiyong Jiang, Rossa W K Chiu
    Abstract:

    BACKGROUND: With the advent of massively parallel sequencing (MPS), DNA analysis can now be performed in a genomewide manner. Recent studies have demonstrated the high precision of MPS for quantifying fetal DNA in Maternal Plasma. In addition, paired-end sequencing can be used to determine the size of each sequenced DNA fragment. We applied MPS in a high-resolution investigation of the clearance profile of circulating fetal DNA. METHODS: Using paired-end MPS, we analyzed serial samples of Maternal Plasma collected from 13 women after cesarean delivery. We also studied the transrenal excretion of circulating fetal DNA in 3 of these individuals by analyzing serial urine samples collected after delivery. RESULTS: The clearance of circulating fetal DNA occurred in 2 phases, with different kinetics. The initial rapid phase had a mean half-life of approximately 1 h, whereas the subsequent slow phase had a mean half-life of approximately 13 h. The final disappearance of circulating fetal DNA occurred at about 1 to 2 days postpartum. Although transrenal excretion was involved in the clearance of circulating fetal DNA, it was not the major route. Furthermore, we observed significant changes in the size profiles of circulating Maternal DNA after delivery, but we did not observe such changes in circulating fetal DNA. CONCLUSIONS: MPS of Maternal Plasma and urinary DNA permits high-resolution study of the clearance profile of circulating fetal DNA.

  • noninvasive prenatal determination of twin zygosity by Maternal Plasma dna analysis
    Clinical Chemistry, 2013
    Co-Authors: Tak Yeung Leung, Rossa W K Chiu, Hao Sun, Peiyong Jiang, Gary J W Liao, Yvonne Kwun Yue Cheng, K Allen C Chan
    Abstract:

    BACKGROUND: The current methods for distinguishing the zygosities of twins include ultrasound scanning, which is nondefinitive, and amniocentesis, which is invasive. We explored the use of massively parallel sequencing of Maternal Plasma DNA for the noninvasive prenatal assessment of the zygosities of twin pregnancies. METHODS: Plasma DNA was extracted from blood collected from 8 women pregnant with twins. Target enrichment and massively parallel sequencing were performed for each Plasma DNA library. Apparent fractional fetal DNA concentrations were calculated for multiple genomic regions by determining the ratio of minor to major alleles among single-nucleotide polymorphism sites. Variations in the apparent fractional fetal DNA concentrations between genomic regions were used to infer whether individual fetuses in a twin pair were genotypically different and hence dizygotic. RESULTS: The extent of the variation in the apparent fractional fetal DNA concentration across chromosomes was 0.82–1.35 SDs for monozygotic twin pregnancies and 2.42–4.80 SDs for dizygotic twin pregnancies. The proportions of apparent fractional fetal DNA concentration values that deviated beyond the range expected for stochastic variation were 0.00%–1.93% for monozygotic twin pregnancies and 36.2%–78.1% for dizygotic twin pregnancies. After identifying a pair of twins as likely dizygotic, the method also allowed determination of the fractional fetal DNA concentrations contributed by the individual fetuses of a dizygotic twin pair. CONCLUSIONS: Noninvasive prenatal determination of twin zygosity by Maternal Plasma DNA sequencing is feasible. It is also possible to determine the relative fractional fetal DNA concentrations for each fetus for dizygotic twin pregnancies.

  • noninvasive prenatal diagnosis of monogenic diseases by targeted massively parallel sequencing of Maternal Plasma application to β thalassemia
    Clinical Chemistry, 2012
    Co-Authors: Peiyong Jiang, K Allen C Chan, Rossa W K Chiu, Tak Yeung Leung, Gary J W Liao, Y Dennis M Lo
    Abstract:

    BACKGROUND: A genomewide genetic and mutational profile of a fetus was recently determined via deep sequencing of Maternal Plasma DNA. This technology could have important applications for noninvasive prenatal diagnosis (NIPD) of many monogenic diseases. Relative haplotype dosage (RHDO) analysis, a core step of this procedure, would allow one to elucidate the Maternally inherited half of the fetal genome. For clinical applications, the cost and complexity of data analysis might be reduced via targeted application of this approach to selected genomic regions containing disease-causing genes. There is thus a need to explore the feasibility of performing RHDO analysis in a targeted manner. METHODS: We performed target enrichment by using solution-phase hybridization followed by massively parallel sequencing of the β-globin gene region in 2 families undergoing prenatal diagnosis for β-thalassemia. We used digital PCR strategies to physically deduce parental haplotypes. Finally, we performed RHDO analysis with target-enriched sequencing data and parental haplotypes to reveal the β-thalassemic status for the fetuses. RESULTS: A mean sequencing depth of 206-fold was achieved in the β-globin gene region by targeted sequencing of Maternal Plasma DNA. RHDO analysis was successful for the sequencing data obtained from the target-enriched samples, including a region in one of the families in which the parents had similar haplotype structures. Data analysis revealed that both fetuses were heterozygous carriers of β-thalassemia. CONCLUSIONS: Targeted sequencing of Maternal Plasma DNA for NIPD of monogenic diseases is feasible.

Tak Yeung Leung - One of the best experts on this subject based on the ideXlab platform.

  • noninvasive prenatal methylomic analysis by genomewide bisulfite sequencing of Maternal Plasma dna
    Clinical Chemistry, 2013
    Co-Authors: Fiona M F Lun, K Allen C Chan, Rossa W K Chiu, Tak Yeung Leung, Kun Sun, Peiyong Jiang, Hao Sun
    Abstract:

    BACKGROUND: Epigenetic mechanisms play an important role in prenatal development, but fetal tissues are not readily accessible. Fetal DNA molecules are present in Maternal Plasma and can be analyzed noninvasively. METHODS: We applied genomewide bisulfite sequencing via 2 approaches to analyze the methylation profile of Maternal Plasma DNA at single-nucleotide resolution. The first approach used Maternal blood samples and polymorphic differences between the mother and fetus to analyze the fetal methylome across the genome. The second approach used the methylation profile of Maternal blood cells and the fractional fetal DNA concentration in Maternal Plasma to deduce the placental methylomic profile from Maternal Plasma DNA-sequencing data. RESULTS: Because of the noninvasive nature of these approaches, we were able to serially assess the methylation profiles of fetal, placental, and Maternal Plasma with Maternal blood samples collected in the first and third trimesters and after delivery. Gestation-related changes were observed. The fetal methylation profile deduced from Maternal Plasma data resembled that of the placental methylome, both on a genomewide level and per CpG site. Imprinted genes and differentially methylated regions were identified from the Maternal Plasma data. We demonstrated one potential clinical application of Maternal Plasma bisulfite sequencing with the successful detection of fetal trisomy 21. CONCLUSIONS: We successfully analyzed fetal and placental methylomes on a genomewide scale, noninvasively and serially. This development offers a powerful method for research, biomarker discovery, and clinical testing for pregnancy-related disorders.

  • high resolution profiling of fetal dna clearance from Maternal Plasma by massively parallel sequencing
    Clinical Chemistry, 2013
    Co-Authors: Shara W Y Lee, K Allen C Chan, Tak Yeung Leung, Peiyong Jiang, Rossa W K Chiu
    Abstract:

    BACKGROUND: With the advent of massively parallel sequencing (MPS), DNA analysis can now be performed in a genomewide manner. Recent studies have demonstrated the high precision of MPS for quantifying fetal DNA in Maternal Plasma. In addition, paired-end sequencing can be used to determine the size of each sequenced DNA fragment. We applied MPS in a high-resolution investigation of the clearance profile of circulating fetal DNA. METHODS: Using paired-end MPS, we analyzed serial samples of Maternal Plasma collected from 13 women after cesarean delivery. We also studied the transrenal excretion of circulating fetal DNA in 3 of these individuals by analyzing serial urine samples collected after delivery. RESULTS: The clearance of circulating fetal DNA occurred in 2 phases, with different kinetics. The initial rapid phase had a mean half-life of approximately 1 h, whereas the subsequent slow phase had a mean half-life of approximately 13 h. The final disappearance of circulating fetal DNA occurred at about 1 to 2 days postpartum. Although transrenal excretion was involved in the clearance of circulating fetal DNA, it was not the major route. Furthermore, we observed significant changes in the size profiles of circulating Maternal DNA after delivery, but we did not observe such changes in circulating fetal DNA. CONCLUSIONS: MPS of Maternal Plasma and urinary DNA permits high-resolution study of the clearance profile of circulating fetal DNA.

  • noninvasive prenatal determination of twin zygosity by Maternal Plasma dna analysis
    Clinical Chemistry, 2013
    Co-Authors: Tak Yeung Leung, Rossa W K Chiu, Hao Sun, Peiyong Jiang, Gary J W Liao, Yvonne Kwun Yue Cheng, K Allen C Chan
    Abstract:

    BACKGROUND: The current methods for distinguishing the zygosities of twins include ultrasound scanning, which is nondefinitive, and amniocentesis, which is invasive. We explored the use of massively parallel sequencing of Maternal Plasma DNA for the noninvasive prenatal assessment of the zygosities of twin pregnancies. METHODS: Plasma DNA was extracted from blood collected from 8 women pregnant with twins. Target enrichment and massively parallel sequencing were performed for each Plasma DNA library. Apparent fractional fetal DNA concentrations were calculated for multiple genomic regions by determining the ratio of minor to major alleles among single-nucleotide polymorphism sites. Variations in the apparent fractional fetal DNA concentrations between genomic regions were used to infer whether individual fetuses in a twin pair were genotypically different and hence dizygotic. RESULTS: The extent of the variation in the apparent fractional fetal DNA concentration across chromosomes was 0.82–1.35 SDs for monozygotic twin pregnancies and 2.42–4.80 SDs for dizygotic twin pregnancies. The proportions of apparent fractional fetal DNA concentration values that deviated beyond the range expected for stochastic variation were 0.00%–1.93% for monozygotic twin pregnancies and 36.2%–78.1% for dizygotic twin pregnancies. After identifying a pair of twins as likely dizygotic, the method also allowed determination of the fractional fetal DNA concentrations contributed by the individual fetuses of a dizygotic twin pair. CONCLUSIONS: Noninvasive prenatal determination of twin zygosity by Maternal Plasma DNA sequencing is feasible. It is also possible to determine the relative fractional fetal DNA concentrations for each fetus for dizygotic twin pregnancies.

  • noninvasive prenatal diagnosis of monogenic diseases by targeted massively parallel sequencing of Maternal Plasma application to β thalassemia
    Clinical Chemistry, 2012
    Co-Authors: Peiyong Jiang, K Allen C Chan, Rossa W K Chiu, Tak Yeung Leung, Gary J W Liao, Y Dennis M Lo
    Abstract:

    BACKGROUND: A genomewide genetic and mutational profile of a fetus was recently determined via deep sequencing of Maternal Plasma DNA. This technology could have important applications for noninvasive prenatal diagnosis (NIPD) of many monogenic diseases. Relative haplotype dosage (RHDO) analysis, a core step of this procedure, would allow one to elucidate the Maternally inherited half of the fetal genome. For clinical applications, the cost and complexity of data analysis might be reduced via targeted application of this approach to selected genomic regions containing disease-causing genes. There is thus a need to explore the feasibility of performing RHDO analysis in a targeted manner. METHODS: We performed target enrichment by using solution-phase hybridization followed by massively parallel sequencing of the β-globin gene region in 2 families undergoing prenatal diagnosis for β-thalassemia. We used digital PCR strategies to physically deduce parental haplotypes. Finally, we performed RHDO analysis with target-enriched sequencing data and parental haplotypes to reveal the β-thalassemic status for the fetuses. RESULTS: A mean sequencing depth of 206-fold was achieved in the β-globin gene region by targeted sequencing of Maternal Plasma DNA. RHDO analysis was successful for the sequencing data obtained from the target-enriched samples, including a region in one of the families in which the parents had similar haplotype structures. Data analysis revealed that both fetuses were heterozygous carriers of β-thalassemia. CONCLUSIONS: Targeted sequencing of Maternal Plasma DNA for NIPD of monogenic diseases is feasible.

  • noninvasive prenatal diagnosis of a case of down syndrome due to robertsonian translocation by massively parallel sequencing of Maternal Plasma dna
    Clinical Chemistry, 2011
    Co-Authors: Fiona M F Lun, Tak Yeung Leung, Hao Sun, Tze K Lau, Yoyo Y Jin, Rossa W K Chiu
    Abstract:

    To the Editor: There has been much recent interest in the use of massively parallel sequencing of Maternal Plasma DNA for the detection of fetal Down syndrome, or trisomy 21 (1–4). DNA fragments in Maternal Plasma were sequenced at random to determine if an additional dose of chromosome 21 (chr21)1 sequences was contributed by the fetus. This approach has been shown to be highly robust in distinguishing trisomic and euploid cases. In these studies, however, all recruited trisomy 21 cases possess supernumerary whole chr21, as confirmed by karyotyping. This approach has not been formally shown to be applicable to other forms of the condition. For example, although the ratio of fetal to Maternal DNA in Maternal Plasma has been shown to remain relatively constant across the entire genome for normal chromosomes (5), it is unknown whether an aberrant chromosome (e.g., one containing a chromosomal translocation) would exhibit an atypical genomic representation in the Plasma. As a first step in addressing this issue, we applied the sequencing approach to a case of familial robertsonian translocation. This study was approved by the local research ethics committee. Maternal peripheral blood samples …

Tze K Lau - One of the best experts on this subject based on the ideXlab platform.

  • noninvasive prenatal diagnosis of a case of down syndrome due to robertsonian translocation by massively parallel sequencing of Maternal Plasma dna
    Clinical Chemistry, 2011
    Co-Authors: Fiona M F Lun, Tak Yeung Leung, Hao Sun, Tze K Lau, Yoyo Y Jin, Rossa W K Chiu
    Abstract:

    To the Editor: There has been much recent interest in the use of massively parallel sequencing of Maternal Plasma DNA for the detection of fetal Down syndrome, or trisomy 21 (1–4). DNA fragments in Maternal Plasma were sequenced at random to determine if an additional dose of chromosome 21 (chr21)1 sequences was contributed by the fetus. This approach has been shown to be highly robust in distinguishing trisomic and euploid cases. In these studies, however, all recruited trisomy 21 cases possess supernumerary whole chr21, as confirmed by karyotyping. This approach has not been formally shown to be applicable to other forms of the condition. For example, although the ratio of fetal to Maternal DNA in Maternal Plasma has been shown to remain relatively constant across the entire genome for normal chromosomes (5), it is unknown whether an aberrant chromosome (e.g., one containing a chromosomal translocation) would exhibit an atypical genomic representation in the Plasma. As a first step in addressing this issue, we applied the sequencing approach to a case of familial robertsonian translocation. This study was approved by the local research ethics committee. Maternal peripheral blood samples …

  • noninvasive prenatal diagnosis of hemophilia by microfluidics digital pcr analysis of Maternal Plasma dna
    Blood, 2011
    Co-Authors: Nancy B Y Tsui, K Allen C Chan, Tak Yeung Leung, Tze K Lau, R A Kadir, Claudia Chi, Gillian Mellars, Edward G D Tuddenham, Rossa W K Chiu
    Abstract:

    Hemophilia is a bleeding disorder with X-linked inheritance. Current prenatal diagnostic methods for hemophilia are invasive and pose a risk to the fetus. Cell-free fetal DNA analysis in Maternal Plasma provides a noninvasive mean of assessing fetal sex in such pregnancies. However, the disease status of male fetuses remains unknown if mutation-specific confirmatory analysis is not performed. Here we have developed a noninvasive test to diagnose whether the fetus has inherited a causative mutation for hemophilia from its mother. The strategy is based on a relative mutation dosage approach, which we have previously established for determining the mutational status of fetuses for autosomal disease mutations. In this study, the relative mutation dosage method is used to deduce whether a fetus has inherited a hemophilia mutation on chromosome X by detecting whether the concentration of the mutant or wild-type allele is overrepresented in the Plasma of heterozygous women carrying male fetuses. We correctly detected fetal genotypes for hemophilia mutations in all of the 12 studied Maternal Plasma samples obtained from at-risk pregnancies from as early as the 11th week of gestation. This development would make the decision to undertake prenatal testing less traumatic and safer for at-risk families.

  • Maternal Plasma dna sequencing reveals the genome wide genetic and mutational profile of the fetus
    Science Translational Medicine, 2010
    Co-Authors: K Allen C Chan, Tak Yeung Leung, Hao Sun, Tze K Lau, Fiona M F Lun, Peiyong Jiang, Eric Z Chen, Yama W Zheng, Charles R Cantor, Rossa W K Chiu
    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 detection of fetal trisomy 18 by rna snp allelic ratio analysis using Maternal Plasma serpinb2 mrna a feasibility study
    Prenatal Diagnosis, 2009
    Co-Authors: Nancy B Y Tsui, Tak Yeung Leung, Blenda C K Wong, Tze K Lau, Rossa W K Chiu
    Abstract:

    Objective Non-invasive prenatal diagnosis of chromosome aneuploidies has been achieved by measuring the ratio of two alleles of a single nucleotide polymorphism (SNP) in circulating placental mRNA (the RNA–SNP allelic ratio approach) in Maternal Plasma. We investigated the feasibility of applying this approach for the non-invasive prenatal detection of fetal trisomy 18. Method We targeted serpin peptidase inhibitor, clade B (ovalbumin), membrane 2 (SERPINB2) mRNA, which is transcribed from chromosome 18 and is preferentially expressed by the placenta. We developed a mass-spectrometric assay to measure the SERPINB2 RNA–SNP allelic ratios in the placental samples and Maternal Plasma obtained from pregnancies involving euploid and trisomy 18 fetuses. Results We were able to separate all the euploid and trisomy 18 placentas by their SERPINB2 RNA–SNP allelic ratios. The allelic ratios of the trisomy 18 placentas deviated from the reference interval established from the euploid placentas. Due to the relatively low concentrations of SERPINB2 mRNA in Maternal Plasma, we used pooled Maternal Plasma samples for analysis. We were able to identify three of the four pooled trisomy 18 Plasma samples by their deviated allelic ratios when compared with the reference interval obtained from pooled euploid Plasma samples. Conclusion It is feasible to detect fetal trisomy 18 non-invasively by Maternal Plasma SERPINB2 RNA–SNP analysis provided that sufficient quantities of Plasma samples are used. Copyright © 2009 John Wiley & Sons, Ltd.

  • noninvasive prenatal diagnosis of monogenic diseases by digital size selection and relative mutation dosage on dna in Maternal Plasma
    Proceedings of the National Academy of Sciences of the United States of America, 2008
    Co-Authors: Fiona M F Lun, K Allen C Chan, Tak Yeung Leung, Nancy B Y Tsui, Tze K Lau, Katherine C K Chow, Pimlak Charoenkwan, Chanane Wanapirak, Torpong Sanguansermsri, Charles R Cantor
    Abstract:

    Prenatal diagnosis of monogenic diseases, such as cystic fibrosis and beta-thalassemia, is currently offered as part of public health programs. However, current methods based on chorionic villus sampling and amniocentesis for obtaining fetal genetic material pose a risk to the fetus. Since the discovery of cell-free fetal DNA in Maternal Plasma, the noninvasive prenatal assessment of paternally inherited traits or mutations has been achieved. Due to the presence of background Maternal DNA, which interferes with the analysis of fetal DNA in Maternal Plasma, noninvasive prenatal diagnosis of Maternally inherited mutations has not been possible. Here we describe a digital relative mutation dosage (RMD) approach that determines if the dosages of the mutant and wild-type alleles of a disease-causing gene are balanced or unbalanced in Maternal Plasma. When applied to the testing of women heterozygous for the CD41/42 (-CTTT) and hemoglobin E mutations on HBB, digital RMD allows the fetal genotype to be deduced. The diagnostic performance of digital RMD is dependent on interplay between the fractional fetal DNA concentration and number of DNA molecules in Maternal Plasma. To achieve fetal genotype diagnosis at lower volumes of Maternal Plasma, fetal DNA enrichment is desired. We thus developed a digital nucleic acid size selection (NASS) strategy that effectively enriches the fetal DNA without additional Plasma sampling or experimental time. We show that digital NASS can work in concert with digital RMD to increase the proportion of cases with classifiable fetal genotypes and to bring noninvasive prenatal diagnosis of monogenic diseases closer to reality.

K Allen C Chan - One of the best experts on this subject based on the ideXlab platform.

  • fetal mitochondrial dna in Maternal Plasma in surrogate pregnancies detection and topology
    Prenatal Diagnosis, 2021
    Co-Authors: Maryjane L, K Allen C Chan, Peiyong Jiang, S A Yakovenko, Haiqiang Zhang, Suk Hang Cheng, V P Apryshko, Alex Zhavoronkov, Rossa W K Chiu
    Abstract:

    OBJECTIVES Due to the Maternally-inherited nature of mitochondrial DNA (mtDNA), there is a lack of information regarding fetal mtDNA in the Plasma of pregnant women. We aim to explore the presence and topologic forms of circulating fetal and Maternal mtDNA molecules in surrogate pregnancies. METHODS Genotypic differences between fetal and surrogate Maternal mtDNA were used to identify the fetal and Maternal mtDNA molecules in Plasma. Plasma samples were obtained from the surrogate pregnant mothers. Using cleavage-end signatures of BfaI restriction enzyme, linear and circular mtDNA molecules in Maternal Plasma could be differentiated. RESULTS Fetal-derived mtDNA molecules were mainly linear (median: 88%; range: 80%-96%), whereas approximately half of the Maternal-derived mtDNA molecules were circular (median: 51%; range: 42%-60%). The fetal DNA fraction of linear mtDNA was lower (median absolute difference: 9.8%; range: 1.1%-27%) than that of nuclear DNA (median: 20%; range: 9.7%-35%). The fetal-derived linear mtDNA molecules were shorter than the Maternal-derived ones. CONCLUSION Fetal mtDNA is present in Maternal Plasma, and consists mainly of linear molecules. Surrogate pregnancies represent a valuable clinical scenario for exploring the biology and potential clinical applications of circulating mtDNA, for example, for pregnancies conceived following mitochondrial replacement therapy.

  • noninvasive prenatal methylomic analysis by genomewide bisulfite sequencing of Maternal Plasma dna
    Clinical Chemistry, 2013
    Co-Authors: Fiona M F Lun, K Allen C Chan, Rossa W K Chiu, Tak Yeung Leung, Kun Sun, Peiyong Jiang, Hao Sun
    Abstract:

    BACKGROUND: Epigenetic mechanisms play an important role in prenatal development, but fetal tissues are not readily accessible. Fetal DNA molecules are present in Maternal Plasma and can be analyzed noninvasively. METHODS: We applied genomewide bisulfite sequencing via 2 approaches to analyze the methylation profile of Maternal Plasma DNA at single-nucleotide resolution. The first approach used Maternal blood samples and polymorphic differences between the mother and fetus to analyze the fetal methylome across the genome. The second approach used the methylation profile of Maternal blood cells and the fractional fetal DNA concentration in Maternal Plasma to deduce the placental methylomic profile from Maternal Plasma DNA-sequencing data. RESULTS: Because of the noninvasive nature of these approaches, we were able to serially assess the methylation profiles of fetal, placental, and Maternal Plasma with Maternal blood samples collected in the first and third trimesters and after delivery. Gestation-related changes were observed. The fetal methylation profile deduced from Maternal Plasma data resembled that of the placental methylome, both on a genomewide level and per CpG site. Imprinted genes and differentially methylated regions were identified from the Maternal Plasma data. We demonstrated one potential clinical application of Maternal Plasma bisulfite sequencing with the successful detection of fetal trisomy 21. CONCLUSIONS: We successfully analyzed fetal and placental methylomes on a genomewide scale, noninvasively and serially. This development offers a powerful method for research, biomarker discovery, and clinical testing for pregnancy-related disorders.

  • high resolution profiling of fetal dna clearance from Maternal Plasma by massively parallel sequencing
    Clinical Chemistry, 2013
    Co-Authors: Shara W Y Lee, K Allen C Chan, Tak Yeung Leung, Peiyong Jiang, Rossa W K Chiu
    Abstract:

    BACKGROUND: With the advent of massively parallel sequencing (MPS), DNA analysis can now be performed in a genomewide manner. Recent studies have demonstrated the high precision of MPS for quantifying fetal DNA in Maternal Plasma. In addition, paired-end sequencing can be used to determine the size of each sequenced DNA fragment. We applied MPS in a high-resolution investigation of the clearance profile of circulating fetal DNA. METHODS: Using paired-end MPS, we analyzed serial samples of Maternal Plasma collected from 13 women after cesarean delivery. We also studied the transrenal excretion of circulating fetal DNA in 3 of these individuals by analyzing serial urine samples collected after delivery. RESULTS: The clearance of circulating fetal DNA occurred in 2 phases, with different kinetics. The initial rapid phase had a mean half-life of approximately 1 h, whereas the subsequent slow phase had a mean half-life of approximately 13 h. The final disappearance of circulating fetal DNA occurred at about 1 to 2 days postpartum. Although transrenal excretion was involved in the clearance of circulating fetal DNA, it was not the major route. Furthermore, we observed significant changes in the size profiles of circulating Maternal DNA after delivery, but we did not observe such changes in circulating fetal DNA. CONCLUSIONS: MPS of Maternal Plasma and urinary DNA permits high-resolution study of the clearance profile of circulating fetal DNA.

  • noninvasive prenatal determination of twin zygosity by Maternal Plasma dna analysis
    Clinical Chemistry, 2013
    Co-Authors: Tak Yeung Leung, Rossa W K Chiu, Hao Sun, Peiyong Jiang, Gary J W Liao, Yvonne Kwun Yue Cheng, K Allen C Chan
    Abstract:

    BACKGROUND: The current methods for distinguishing the zygosities of twins include ultrasound scanning, which is nondefinitive, and amniocentesis, which is invasive. We explored the use of massively parallel sequencing of Maternal Plasma DNA for the noninvasive prenatal assessment of the zygosities of twin pregnancies. METHODS: Plasma DNA was extracted from blood collected from 8 women pregnant with twins. Target enrichment and massively parallel sequencing were performed for each Plasma DNA library. Apparent fractional fetal DNA concentrations were calculated for multiple genomic regions by determining the ratio of minor to major alleles among single-nucleotide polymorphism sites. Variations in the apparent fractional fetal DNA concentrations between genomic regions were used to infer whether individual fetuses in a twin pair were genotypically different and hence dizygotic. RESULTS: The extent of the variation in the apparent fractional fetal DNA concentration across chromosomes was 0.82–1.35 SDs for monozygotic twin pregnancies and 2.42–4.80 SDs for dizygotic twin pregnancies. The proportions of apparent fractional fetal DNA concentration values that deviated beyond the range expected for stochastic variation were 0.00%–1.93% for monozygotic twin pregnancies and 36.2%–78.1% for dizygotic twin pregnancies. After identifying a pair of twins as likely dizygotic, the method also allowed determination of the fractional fetal DNA concentrations contributed by the individual fetuses of a dizygotic twin pair. CONCLUSIONS: Noninvasive prenatal determination of twin zygosity by Maternal Plasma DNA sequencing is feasible. It is also possible to determine the relative fractional fetal DNA concentrations for each fetus for dizygotic twin pregnancies.

  • noninvasive prenatal diagnosis of monogenic diseases by targeted massively parallel sequencing of Maternal Plasma application to β thalassemia
    Clinical Chemistry, 2012
    Co-Authors: Peiyong Jiang, K Allen C Chan, Rossa W K Chiu, Tak Yeung Leung, Gary J W Liao, Y Dennis M Lo
    Abstract:

    BACKGROUND: A genomewide genetic and mutational profile of a fetus was recently determined via deep sequencing of Maternal Plasma DNA. This technology could have important applications for noninvasive prenatal diagnosis (NIPD) of many monogenic diseases. Relative haplotype dosage (RHDO) analysis, a core step of this procedure, would allow one to elucidate the Maternally inherited half of the fetal genome. For clinical applications, the cost and complexity of data analysis might be reduced via targeted application of this approach to selected genomic regions containing disease-causing genes. There is thus a need to explore the feasibility of performing RHDO analysis in a targeted manner. METHODS: We performed target enrichment by using solution-phase hybridization followed by massively parallel sequencing of the β-globin gene region in 2 families undergoing prenatal diagnosis for β-thalassemia. We used digital PCR strategies to physically deduce parental haplotypes. Finally, we performed RHDO analysis with target-enriched sequencing data and parental haplotypes to reveal the β-thalassemic status for the fetuses. RESULTS: A mean sequencing depth of 206-fold was achieved in the β-globin gene region by targeted sequencing of Maternal Plasma DNA. RHDO analysis was successful for the sequencing data obtained from the target-enriched samples, including a region in one of the families in which the parents had similar haplotype structures. Data analysis revealed that both fetuses were heterozygous carriers of β-thalassemia. CONCLUSIONS: Targeted sequencing of Maternal Plasma DNA for NIPD of monogenic diseases is feasible.

Tse N Leung - One of the best experts on this subject based on the ideXlab platform.

  • detection of restriction enzyme digested target dna by pcr amplification using a stem loop primer application to the detection of hypomethylated fetal dna in Maternal Plasma
    Clinical Chemistry, 2007
    Co-Authors: Yu K Tong, Chunming Ding, Rossa W K Chiu, Tak Yeung Leung, Tze K Lau, Tse N Leung
    Abstract:

    Background: The discovery of cell-free fetal DNA in Maternal Plasma has opened up new possibilities for noninvasive prenatal diagnosis and monitoring. Among the fetal markers that have been described, methylation markers are sex and polymorphism independent. Methylation-sensitive restriction endonucleases are commonly used to digest hypomethylated DNA molecules, and the hypermethylated molecules remain intact for detection. The positive detection of the cleaved hypomethylated molecules would be useful for certain targets but has not been reported. Methods: The use of a stem-loop primer in microRNA detection has previously been described. In this study, DNA assays were designed and performed on Maternal Plasma, which contained the hypomethylated placental serpin peptidase inhibitor, clade B (ovalbumin), member 5 ( SERPINB5 ; maspin ) gene in an excess background of hypermethylated Maternal SERPINB5 . Detection of the enzyme-digested placenta-derived hypomethylated SERPINB5 molecules was achieved by performing stem-loop extension followed by real-time PCR on Maternal Plasma. The placental origin of the stem-loop–extended SERPINB5 molecules was confirmed by genotyping. Results: From the real-time PCR results on Maternal Plasma, stem-loop–extended SERPINB5 promoter sequences were detectable in all 11 enzyme-digested predelivery Maternal Plasma samples. Postpartum clearance was demonstrated. In 9 cases in which the fetal and Maternal SERPINB5 genotypes were distinguishable, the placental-specific genotypes were detected in all predelivery Maternal Plasma samples. Conclusion: Detection of restriction enzyme-digested hypomethylated placental DNA molecules in Maternal Plasma by the use of a stem-loop primer represents a novel approach in fetal epigenetic marker detection. The analytical approach may also be generally applicable to the detection of restriction enzyme-digested nucleic acid fragments.

  • hypermethylated rassf1a in Maternal Plasma a universal fetal dna marker that improves the reliability of noninvasive prenatal diagnosis
    Clinical Chemistry, 2006
    Co-Authors: K Allen C Chan, Chunming Ding, Ageliki Gerovassili, Sze W Yeung, Tse N Leung, Grace Tinyun Chung, Stephen S.c. Chim, Rossa W K Chiu, Y Dennis M Lo
    Abstract:

    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.

  • noninvasive prenatal detection of fetal trisomy 18 by epigenetic allelic ratio analysis in Maternal Plasma theoretical and empirical considerations
    Clinical Chemistry, 2006
    Co-Authors: Yu Kwan Tong, Chunming Ding, Ageliki Gerovassili, Tse N Leung, Stephen S.c. Chim, Rossa W K Chiu, Tak Yeung Leung, Tze K Lau, K H Nicolaides
    Abstract:

    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.

  • detection of the placental epigenetic signature of the maspin gene in Maternal Plasma
    Proceedings of the National Academy of Sciences of the United States of America, 2005
    Co-Authors: Stephen S.c. Chim, Tse N Leung, Rossa W K Chiu, Cees B M Oudejans, Tze K Lau, Yu Kwan Tong, Lisa Y S Chan, Chunming Ding
    Abstract:

    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.

  • lack of dramatic enrichment of fetal dna in Maternal Plasma by formaldehyde treatment
    Clinical Chemistry, 2005
    Co-Authors: Grace Tinyun Chung, K Allen C Chan, Rossa W K Chiu, Tze K Lau, Tse N Leung
    Abstract:

    The discovery of fetal DNA in Maternal Plasma has opened up new possibilities for noninvasive prenatal diagnosis (1)(2)(3)(4)(5)(6). A recent report(7) indicated that the proportion of fetal DNA in Maternal Plasma can be dramatically enriched through the adoption of a blood-processing protocol involving the addition of formaldehyde to Maternal blood samples. Dhallan et al. (7) suggested that these observations might be the result of several factors, including a reduction in background Maternal DNA by minimization of Maternal cell lysis through formaldehyde-mediated cell membrane stabilization and the use of a gentle centrifugation protocol, as well as the preservation of fetal DNA through nuclease inhibition by formaldehyde. In view of the profound implications of the study (7), we aimed to validate and investigate the underlying mechanisms of the reported phenomenon. To assess the effects of several contributory factors, our study was conducted in three successive stages. In the first part of the study, we aimed to verify the effects of the previously published protocol (7) on total DNA concentrations in Plasma from nonpregnant individuals. In the second part of the study, we evaluated the effects of formaldehyde addition on total and fetal DNA concentrations in Maternal Plasma in relation to the time of blood processing (0, 6, and 24 h after blood collection). In the last part of the study, we investigated whether the reported enrichment in circulating fetal DNA concentrations (7) might be a consequence of the imprecision of the analytical method chosen by the authors. All participants were recruited with informed consent from the Prince of Wales Hospital, Hong Kong, with institutional ethics approval. In the first part of the study, blood (24 mL) was collected into 3-mL EDTA tubes from each of eight nonpregnant volunteers and divided into …