Peter Tsang

University of Washington Seattle, Seattle, WA, United States

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Publications (13)160.79 Total impact

  • Cancer Research 08/2013; 73(8 Supplement):4205-4205. · 8.65 Impact Factor
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    ABSTRACT: Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are oppositely imprinted autism-spectrum disorders with known genetic bases, but complex epigenetic mechanisms underlie their pathogenesis. The PWS/AS locus on 15q11-q13 is regulated by an imprinting control region that is maternally methylated and silenced. The PWS imprinting control region is the promoter for a one megabase paternal transcript encoding the ubiquitous protein-coding Snrpn gene and multiple neuron-specific noncoding RNAs, including the PWS-related Snord116 repetitive locus of small nucleolar RNAs and host genes, and the antisense transcript to AS-causing ubiquitin ligase encoding Ube3a (Ube3a-ATS). Neuron-specific transcriptional progression through Ube3a-ATS correlates with paternal Ube3a silencing and chromatin decondensation. Interestingly, topoisomerase inhibitors, including topotecan, were recently identified in an unbiased drug screen for compounds that could reverse the silent paternal allele of Ube3a in neurons, but the mechanism of topotecan action on the PWS/AS locus is unknown. Here, we demonstrate that topotecan treatment stabilizes the formation of RNA:DNA hybrids (R loops) at G-skewed repeat elements within paternal Snord116, corresponding to increased chromatin decondensation and inhibition of Ube3a-ATS expression. Neural precursor cells from paternal Snord116 deletion mice exhibit increased Ube3a-ATS levels in differentiated neurons and show a reduced effect of topotecan compared with wild-type neurons. These results demonstrate that the AS candidate drug topotecan acts predominantly through stabilizing R loops and chromatin decondensation at the paternally expressed PWS Snord116 locus. Our study holds promise for targeted therapies to the Snord116 locus for both AS and PWS.
    Proceedings of the National Academy of Sciences 08/2013; · 9.81 Impact Factor
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    ABSTRACT: Prader-Willi syndrome (PWS), a genetic disorder of obesity, intellectual disability and sleep abnormalities, is caused by loss of noncoding RNAs on paternal chromosome 15q11-q13. The imprinted minimal PWS locus encompasses a long non-coding RNA (lncRNA) transcript processed into multiple SNORD116 small nucleolar RNAs (snoRNAs) and the spliced exons of the host gene, 116HG. However, both the molecular function and disease relevance of the spliced lncRNA 116HG are unknown. Here we show that 116HG forms a subnuclear RNA cloud that co-purifies with the transcriptional activator RBBP5 and active metabolic genes, remains tethered to the site of its transcription, and increases in size in postnatal neurons and during sleep. Snord116del mice lacking 116HG exhibited increased energy expenditure corresponding to dysregulation of diurnally expressed Mtor and circadian genes Clock, Cry1, and Per2. These combined genomic and metabolic analyses demonstrate that 116HG regulates diurnal energy expenditure of the brain. These novel molecular insights into the energy imbalance in PWS should lead to improved therapies and understanding of lncRNA roles in complex neurodevelopmental and metabolic disorders.
    Human Molecular Genetics 06/2013; · 7.69 Impact Factor
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    ABSTRACT: Female human induced pluripotent stem cell (hiPSC) lines exhibit variability in X-inactivation status. The majority of hiPSC lines maintain one transcriptionally active X (Xa) and one inactive X (Xi) chromosome from donor cells. However, at low frequency, hiPSC lines with two Xas are produced, suggesting that epigenetic alterations of the Xi occur sporadically during reprogramming. We show here that X-inactivation status in female hiPSC lines depends on derivation conditions. hiPSC lines generated by the Kyoto method (retroviral or episomal reprogramming), which uses leukemia inhibitory factor (LIF)-expressing SNL feeders, frequently had two Xas. Early passage Xa/Xi hiPSC lines generated on non-SNL feeders were converted into Xa/Xa hiPSC lines after several passages on SNL feeders, and supplementation with recombinant LIF caused reactivation of some of X-linked genes. Thus, feeders are a significant factor affecting X-inactivation status. The efficient production of Xa/Xa hiPSC lines provides unprecedented opportunities to understand human X-reactivation and -inactivation.
    Cell stem cell 07/2012; 11(1):91-9. · 23.56 Impact Factor
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    ABSTRACT: Copy-number variants (CNVs) can reach appreciable frequencies in the human population, and recent discoveries have shown that several of these copy-number polymorphisms (CNPs) are associated with human diseases, including lupus, psoriasis, Crohn disease, and obesity. Despite new advances, significant biases remain in terms of CNP discovery and genotyping. We developed a method based on single-channel intensity data and benchmarked against copy numbers determined from sequencing read depth to successfully obtain CNP genotypes for 1495 CNPs from 487 human DNA samples of diverse ethnic backgrounds. This microarray contained CNPs in segmental duplication-rich regions and insertions of sequences not represented in the reference genome assembly or on standard SNP microarray platforms. We observe that CNPs in segmental duplications are more likely to be population differentiated than CNPs in unique regions (p = 0.015) and that biallelic CNPs show greater stratification when compared to frequency-matched SNPs (p = 0.0026). Although biallelic CNPs show a strong correlation of copy number with flanking SNP genotypes, the majority of multicopy CNPs do not (40% with r > 0.8). We selected a subset of CNPs for further characterization in 1876 additional samples from 62 populations; this revealed striking population-differentiated structural variants in genes of clinical significance such as OCLN, a tight junction protein involved in hepatitis C viral entry. Our microarray design allows these variants to be rapidly tested for disease association and our results suggest that CNPs (especially those that cannot be imputed from SNP genotypes) might have contributed disproportionately to human diversity and selection.
    The American Journal of Human Genetics 03/2011; 88(3):317-32. · 11.20 Impact Factor
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    ABSTRACT: The discovery of complex structural variations that exist within individual genomes has prompted a need to visualize chromosomes at a higher resolution than previously possible. To address this concern, we established a robust, high-resolution fluorescence in situ hybridization (FISH) method that utilizes probes derived from high complexity libraries of long oligonucleotides (>150 mers) synthesized in massively parallel reactions. In silico selected oligonucleotides, targeted to only the most informative elements in 18 genomic regions of interest, eliminated the need for suppressive hybridization reagents. Because of the inherent flexibility in our probe design methods, we readily visualized regions as small as 6.7 kb with high specificity on human metaphase chromosomes, resulting in an overall success rate of 94%. Two-color FISH over a 479-kb duplication, initially reported as being identical in 2 individuals, revealed distinct 2-color patterns representing direct and inverted duplicons, demonstrating that visualization by high-resolution FISH provides further insight in the fine-scale complexity of genomic structures. The ability to design FISH probes for any sequenced genome along with the ease, reproducibility, and high level of accuracy of this technique suggests that it will be powerful for routine analysis of previously difficult genomic regions and structures.
    Cytogenetic and Genome Research 01/2011; 132(4):248-54. · 1.84 Impact Factor
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    ABSTRACT: The extent of human genomic structural variation suggests that there must be portions of the genome yet to be discovered, annotated and characterized at the sequence level. We present a resource and analysis of 2,363 new insertion sequences corresponding to 720 genomic loci. We found that a substantial fraction of these sequences are either missing, fragmented or misassigned when compared to recent de novo sequence assemblies from short-read next-generation sequence data. We determined that 18-37% of these new insertions are copy-number polymorphic, including loci that show extensive population stratification among Europeans, Asians and Africans. Complete sequencing of 156 of these insertions identified new exons and conserved noncoding sequences not yet represented in the reference genome. We developed a method to accurately genotype these new insertions by mapping next-generation sequencing datasets to the breakpoint, thereby providing a means to characterize copy-number status for regions previously inaccessible to single-nucleotide polymorphism microarrays.
    Nature Methods 05/2010; 7(5):365-71. · 23.57 Impact Factor
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    ABSTRACT: Genetic variation among individual humans occurs on many different scales, ranging from gross alterations in the human karyotype to single nucleotide changes. Here we explore variation on an intermediate scale—particularly insertions, deletions and inversions affecting from a few thousand to a few million base pairs. We employed a clone-based method to interrogate this intermediate structural variation in eight individuals of diverse geographic ancestry. Our analysis provides a comprehensive overview of the normal pattern of structural variation present in these genomes, refining the location of 1,695 structural variants. We find that 50% were seen in more than one individual and that nearly half lay outside regions of the genome previously described as structurally variant. We discover 525 new insertion sequences that are not present in the human reference genome and show that many of these are variable in copy number between individuals. Complete sequencing of 261 structural variants reveals considerable locus complexity and provides insights into the different mutational processes that have shaped the human genome. These data provide the first high-resolution sequence map of human structural variation—a standard for genotyping platforms and a prelude to future individual genome sequencing projects.
    Nature 04/2008; 453(7191):56-64. · 38.60 Impact Factor
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    ABSTRACT: Despite considerable excitement over the potential functional significance of copy-number variants (CNVs), we still lack knowledge of the fine-scale architecture of the large majority of CNV regions in the human genome. In this study, we used a high-resolution array-based comparative genomic hybridization (aCGH) platform that targeted known CNV regions of the human genome at approximately 1 kb resolution to interrogate the genomic DNAs of 30 individuals from four HapMap populations. Our results revealed that 1020 of 1153 CNV loci (88%) were actually smaller in size than what is recorded in the Database of Genomic Variants based on previously published studies. A reduction in size of more than 50% was observed for 876 CNV regions (76%). We conclude that the total genomic content of currently known common human CNVs is likely smaller than previously thought. In addition, approximately 8% of the CNV regions observed in multiple individuals exhibited genomic architectural complexity in the form of smaller CNVs within larger ones and CNVs with interindividual variation in breakpoints. Future association studies that aim to capture the potential influences of CNVs on disease phenotypes will need to consider how to best ascertain this previously uncharacterized complexity.
    The American Journal of Human Genetics 04/2008; 82(3):685-95. · 11.20 Impact Factor
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    ABSTRACT: A database of apparently benign copy number variants (bCNVs) detected by a Spectral Genomics Inc./PerkinElmer BAC array platform has been maintained through the University of Utah Comparative Genomic Hybridization laboratory since 2005. The target population for this database represents 1,275 patients with abnormal phenotypes, primarily children referred for developmental delay and mental retardation. These bCNVs are independent of any identified copy number abnormality detected. The most common 35 bCNVs observed and their frequencies are reported here, and a subset of ten of the patients studied was evaluated on a new oligonucleotide CNV array set designed by Agilent Technologies. There was a 76% concordance of calls for these 35 bCNVs detected by both array platforms in the same patients. The higher resolution of the Agilent oligonucleotide array compared to the BAC array allowed determination of the precise breakpoints of the observed CNVs, in addition to documentation of additional CNVs of smaller sizes. As expected, observed CNVs and their frequencies were generally consistent with those of other previously published and available databases, including the Database of Genomic Variants (http://projects.tcag.ca/variation/). The availability of these data should assist other clinical laboratories in the evaluation of CNVs of unknown clinical significance.
    Cytogenetic and Genome Research 02/2008; 123(1-4):94-101. · 1.84 Impact Factor
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    ABSTRACT: The discovery of copy number variation in healthy individuals is far from complete, and owing to the resolution of detection systems used, the majority of loci reported so far are relatively large ( approximately 65%>10 kb). Applying a two-stage high-resolution array comparative genomic hybridization approach to analyse 50 healthy Caucasian males from northern France, we discovered 2208 copy number variants (CNVs) detected by more than one consecutive probe. These clustered into 1469 CNV regions (CNVRs), of which 721 are thought to be novel. The majority of these are small (median size 4.4 kb) and most have common boundaries, with a coefficient of variation less than 0.1 for 83% of endpoints in those observed in multiple samples. Only 6% of the CNVRs analysed showed evidence of both copy number losses and gains at the same site. A further 6089 variants were detected by single probes: 48% of these were observed in more than one individual. In total, 2570 genes were seen to intersect variants: 1284 in novel loci. Genes involved in differentiation and development were significantly over-represented and approximately half of the genes identified feature in the Online Mendelian Inheritance in Man database. The biological importance of many genes affected, along with the well-conserved nature of the majority of the CNVs, suggests that they could have important implications for phenotype and, thus, be useful for association studies of complex diseases.
    Human Molecular Genetics 12/2007; 16(23):2783-94. · 7.69 Impact Factor
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    ABSTRACT: Array-based comparative genomic hybridization (aCGH) measures copy number variations at multiple loci simultaneously, providing an important tool for studying genomic alterations associated with cancer, developmental disorders, and germline copy number polymorphisms. The broadest utility of aCGH is obtained by enabling flexible and high-resolution probing of regions of interest while preserving the greatest possible complexity of targets derived from whole genome samples. We therefore developed probe design criteria, assay conditions, and analysis methods that enable 60-mer oligonucleotide arrays to be used for CGH measurements using total genomic DNA [1]. We designed a 60-mer oligonucleotide array with 40K probes specifically designed for CGH representing sequences throughout the human genome with a bias for known and predicted gene loci. We tested the performance of this array for reproducibly measuring and mapping losses, and amplification events of varying levels and sizes using both unamplified and phi29 (Qiagen, Valenica, CA, USA) amplified total genomic DNA from a series of model systems. The mean slope of experimental versus theoretical log-ratios for chromosome X probes on this genome-wide human CGH array in XY versus XX hybridizations typically exceeds 0.9, with probe by probe error rates of less than 10% in the separation of their log-ratio distributions. Additionally, we used this platform to examine well-characterized cell lines, including diploid cells with partial deletions in chromosome 18q, and diploid and aneuploid tumor cell lines with known amplification and deletion events. We show that the highly processive DNA polymerase phi29 can be used to prepare aCGH templates from as little as 10 ng starting material that yield high-quality aCGH measurements throughout the genome. While phi29 provides a simplified isothermal method for amplifying limiting material, non-specific DNA fragments of high MW are generated in the absence of sufficient input template. Although these products do not hybridize to the array, the presence of these amplification products obscures the accurate quantification of DNA template specific to the input genomic DNA prior to the labeling reaction. To ensure reproducible and robust aCGH assay quality, we developed methods and protocols using the Agilent BioAnalyzer (Agilent Technologies, Palo Alto, CA, USA) to enable accurate quality control for key prehybridization steps, including: phi29 amplification of genomic samples, restriction digestion of templates and target labeling. We have also developed visualization tools and statistically robust computational tools that take into account the estimated errors on the measured log ratios in mapping aberration boundaries, and for identifying common aberrations across multiple samples. We tested the reproducibility of our platform using tumor cell line samples including the colon adenocarcinoma cell line HT29 in hybridizations performed in different laboratories (Agilent Labs, National Human Genome Research Institute, Translational Genomics Institute). We present results, using these methods, demonstrating that in situ synthesized 60-mer oligonucleotide arrays can reproducibly detect genomic lesions including single copy and homozygous deletions, and variable amplicons throughout the genome using full complexity genomic DNA samples.
    Breast Cancer Research 06/2005; 7:1-1. · 5.33 Impact Factor
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    ABSTRACT: Array-based comparative genomic hybridization (CGH) measures copy-number variations at multiple loci simultaneously, providing an important tool for studying cancer and developmental disorders and for developing diagnostic and therapeutic targets. Arrays for CGH based on PCR products representing assemblies of BAC or cDNA clones typically require maintenance, propagation, replication, and verification of large clone sets. Furthermore, it is difficult to control the specificity of the hybridization to the complex sequences that are present in each feature of such arrays. To develop a more robust and flexible platform, we created probe-design methods and assay protocols that make oligonucleotide microarrays synthesized in situ by inkjet technology compatible with array-based comparative genomic hybridization applications employing samples of total genomic DNA. Hybridization of a series of cell lines with variable numbers of X chromosomes to arrays designed for CGH measurements gave median ratios for X-chromosome probes within 6% of the theoretical values (0.5 for XY/XX, 1.0 for XX/XX, 1.4 for XXX/XX, 2.1 for XXXX/XX, and 2.6 for XXXXX/XX). Furthermore, these arrays detected and mapped regions of single-copy losses, homozygous deletions, and amplicons of various sizes in different model systems, including diploid cells with a chromosomal breakpoint that has been mapped and sequenced to a precise nucleotide and tumor cell lines with highly variable regions of gains and losses. Our results demonstrate that oligonucleotide arrays designed for CGH provide a robust and precise platform for detecting chromosomal alterations throughout a genome with high sensitivity even when using full-complexity genomic samples.
    Proceedings of the National Academy of Sciences 01/2005; 101(51):17765-70. · 9.81 Impact Factor

Publication Stats

1k Citations
160.79 Total Impact Points

Institutions

  • 2010–2011
    • University of Washington Seattle
      • Department of Genome Sciences
      Seattle, WA, United States
  • 2008
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States
    • Agilent Technologies
      Santa Clara, California, United States