
Georges NatsoulisStanford University | SU · Stanford Genome Technology Center
Georges Natsoulis
Doctor of Engineering
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67
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Publications (67)
The myeloproliferative neoplasms (MPN) are a group of clonal hematopoietic stem cell disorders that include essential thrombocythemia (ET), polycythemia vera (PV) and myelofibrosis (MF) which arise from somatic mutations in JAK2, MPL or CALR, but which can evolve to acute myeloid leukemia (AML) by the acquisition of additional somatic mutations.
To...
The ultimate goal in the treatment of ET is to eliminate mutant cells with the capacity to both proliferate and self-renew. Lysine-specific demethylase-1, LSD1, is an enzyme critical for regulating the proliferation of hematopoietic stem cells and the maturation of progenitors (Sprussel et al. 2012). Bomedemstat is an orally active LSD1 inhibitor t...
Many patients with ET are resistant to or intolerant of current standards of care (SOC) - hydroxyurea (HU), interferon, anagrelide - underscoring the need for novel therapies with distinct modes of action that reduce the risk of thrombosis, improve the patient's experience and favorably alter the natural history. Lysine-specific demethylase-1 (LSD1...
There is an unmet need for novel therapies with distinct modes of action to offer clinical benefit for patients with myelofibrosis (MF) who become resistant or intolerant to JAK inhibitors. Lysine-specific demethylase-1 (LSD1) is a histone demethylase critical for self-renewal potential of malignant myeloid cells for hematopoietic differentiation,...
First reported in 1999, germline runt-related transcription factor 1 (RUNX1) mutations are a well-established cause of familial platelet disorder with predisposition to myeloid malignancy (FPD-MM). We present the clinical phenotypes and genetic mutations detected in 10 novel RUNX1-mutated FPD-MM families. Genomic analyses on these families detected...
Ruxolitinib (Jakafi®) is the one approved therapy for myelofibrosis (MF) based on reduction of splenomegaly and symptoms but JAK inhibition has not proven to significantly modify disease progression. There remains the need for novel therapies with distinct modes of action that can improve the patient experience of MF and impact progression. Lysine-...
Background: It has been known for approximately 19 years that germline mutations in RUNX1, lead to familial platelet disorder with predisposition to myeloid malignancy (FPD-MM, OMIM 601399). Since that time researchers have identified a broad range of different RUNX1 mutations, in over 100 families. In large families, the diagnosis of malignancy sh...
Background: One of the more important prognostic factors used to predict the outcome in acute myeloid leukemia (AML) is the persistence of leukemic cells after treatment. The reliable measurement of residual disease (MRD) offers many other clinical uses besides. An assay that was facile, affordable, and applicable to the broadest group of patients...
Gastric cancer is the second-leading cause of global cancer deaths, with metastatic disease representing the primary cause of mortality. To identify candidate drivers involved in oncogenesis and tumor evolution, we conduct an extensive genome sequencing analysis of metastatic progression in a diffuse gastric cancer. This involves a comparison betwe...
We have developed a targeted resequencing approach referred to as Oligonucleotide-Selective Sequencing. In this study, we
report a series of significant improvements and novel applications of this method whereby the surface of a sequencing flow
cell is modified in situ to capture specific genomic regions of interest from a sample and then sequenced...
Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC
We conducted a detailed genetic and functional analysis of personalized tumor evolution in a case of diffuse gastric cancer. Leveraging the unique genetics of an individual with a confirmed germline mutation in the causative CDH1 tumor suppressor gene, we identified candid...
Somatic mutations, chromosomal rearrangements and other genomic aberrations in cancer genomes are critical events in the development of cancer and increasingly used in identifying oncogenic events that may be indicators for targeted therapies. For rapid and high accuracy analysis of targeted candidate genes and regions from cancer genomes, we devel...
New cancer therapies are increasingly geared towards exploiting critical genetic and genomic features specific to the tumor. These mutations and genomic aberrations are the basis for precision cancer medicine. Thus, rapid molecular characterization of clinical cancer samples has become increasingly important for cancer targeted therapy development....
Taking advantage of the deep targeted sequencing capabilities of next generation sequencers, we have developed a novel two step insertion deletion (indel) detection algorithm (IDA) that can determine indels from single read sequences with high computational efficiency and sensitivity when indels are fractionally less compared to wild type reference...
We develop statistically based methods to detect single nucleotide DNA
mutations in next generation sequencing data. Sequencing generates counts of
the number of times each base was observed at hundreds of thousands to billions
of genome positions in each sample. Using these counts to detect mutations is
challenging because mutations may have very...
Whole-genome sequencing is becoming commonplace, but the accuracy and completeness of variant calling by the most widely used platforms from Illumina and Complete Genomics have not been reported. Here we sequenced the genome of an individual with both technologies to a high average coverage of ~76×, and compared their performance with respect to se...
With next-generation DNA sequencing technologies, one can interrogate a specific genomic region of interest at very high depth
of coverage and identify less prevalent, rare mutations in heterogeneous clinical samples. However, the mutation detection
levels are limited by the error rate of the sequencing technology as well as by the availability of...
Whole-genome sequencing is becoming commonplace, but the accuracy and completeness of variant calling by the most widely used platforms from Illumina and Complete Genomics have not been reported. Here we sequenced the genome of an individual with both technologies to a high average coverage of ∼76×, and compared their performance with respect to se...
Molecular steps in targeted circular sequencing libraries. This file contains a detailed DNA sequences and molecular biology descriptions of the targeted library assay.
Oligonucleotide sequences. This file includes the sequences and molecular properties of the capture oligonucleotides.
For next generation DNA sequencing, we have developed a rapid and simple approach for preparing DNA libraries of targeted DNA content. Current protocols for preparing DNA for next-generation targeted sequencing are labor-intensive, require large amounts of starting material, and are prone to artifacts that result from necessary PCR amplification of...
Recent exponential growth in the throughput of next-generation DNA sequencing platforms has dramatically spurred the use of
accessible and scalable targeted resequencing approaches. This includes candidate region diagnostic resequencing and novel
variant validation from whole genome or exome sequencing analysis. We have previously demonstrated that...
Highly multiplex DNA sequencers have greatly expanded our ability to survey human genomes for previously unknown single nucleotide
polymorphisms (SNPs). However, sequencing and mapping errors, though rare, contribute substantially to the number of false
discoveries in current SNP callers. We demonstrate that we can significantly reduce the number o...
We describe an approach for targeted genome resequencing, called oligonucleotide-selective sequencing (OS-Seq), in which we modify the immobilized lawn of oligonucleotide primers of a next-generation DNA sequencer to function as both a capture and sequencing substrate. We apply OS-Seq to resequence the exons of either 10 or 344 cancer genes from hu...
Supporting tables and description of the cost assessment.
(DOC)
The selective genomic circularization process. Genomic DNA is digested with one of several possible restriction enzymes. The restriction digest is mixed with a pool of targeting oligonucleotides and a single 40 base oligonucleotide vector. Each targeting oligonucleotide has two 20 base capture arms complementary to genomic DNA. One of the capture a...
We have developed an integrated strategy for targeted resequencing and analysis of gene subsets from the human exome for variants. Our capture technology is geared towards resequencing gene subsets substantially larger than can be done efficiently with simplex or multiplex PCR but smaller in scale than exome sequencing. We describe all the steps fr...
315
Introduction
Dysregulated JAK-STAT signaling in chronic myeloproliferative neoplasms (MPNs) has primarily been attributed to activating mutations in tyrosine kinases. However, JAK-STAT activation can be demonstrated in some patients lacking JAK2 or MPL mutations, suggesting alteration of other regulatory elements in this pathway. One regulator...
Dysregulated JAK-STAT signaling is a hallmark of myeloproliferative neoplasms (MPNs), as evidenced by the identification of activating mutations in JAK2, and the thrombopoietin (TPO) receptor MPL in a subset of MPN patients. Clinical trials with highly specific inhibitors of JAK2 are currently ongoing, and clinical responses have been observed in t...
Supplementary Material 1
Supplementary Information
Supplementary Material 2
We have used a supervised classification approach to systematically mine a large microarray database derived from livers of compound-treated rats. Thirty-four distinct signatures (classifiers) for pharmacological and toxicological end points can be identified. Just 200 genes are sufficient to classify these end points. Signatures were enriched in x...
We have assembled a large toxicogenomic database, DrugMatrix®, containing microarray expression profiles from short-term repeat dose rat studies for over 630 reference drugs and toxicants. Gene expression profiles were collected from up to seven different tissues, in addition to standard hematology, clinical chemistry, histopathology and pharmacolo...
We consider the problem of fitting a large-scale covariance matrix to multivariate Gaussian data in such a way that the inverse is sparse, thus providing model selection. Beginning with a dense empirical covariance matrix, we solve a maximum likelihood problem with an l1-norm penalty term added to encourage sparsity in the inverse. For models with...
Successful drug discovery requires accurate decision making in order to advance the best candidates from initial lead identification to final approval. Chemogenomics, the use of genomic tools in pharmacology and toxicology, offers a promising enhancement to traditional methods of target identification/validation, lead identification, efficacy evalu...
A large gene expression database has been produced that characterizes the gene expression and physiological effects of hundreds of approved and withdrawn drugs, toxicants, and biochemical standards in various organs of live rats. In order to derive useful biological knowledge from this large database, a variety of supervised classification algorith...
One application of genomics in drug safety assessment is the identification of biomarkers to predict compound toxicity before it is detected using traditional approaches, such as histopathology. However, many genomic approaches have failed to demonstrate superiority to traditional methods, have not been appropriately validated on external samples,...
We consider a binary, linear classification problem in which the data points are assumed to be unknown, but bounded within given hyper-rectangles, i.e., the covariates are bounded within intervals explicitly given for each data point separately. We address the problem of designing a robust classifier in this setting by minimizing the worst-case val...
Compounds that selectively disrupt fungal mitosis have proven to be effective in controlling agricultural pests, but no specific
mitotic inhibitor is available for the treatment of systemic mycoses in mammalian hosts. In an effort to identify novel mitotic
inhibitors, we used a cell-based screening strategy that exploited the hypersensitivity of a...
Improved lead prioritization in relation to toxicity, efficacy and mechanism of action could improve the overall success of drug discovery. DrugMatrix™ is an integrated database incorporating compound data including molecular pharmacology, gene expression, chemical structure and SAR, toxicology, pathology, and pharmacology. This database helps drug...
Although genomics tools and approaches abound, and many new genes and proteins have been identified, genomics has not contributed very effectively to improving the efficiencies and success rates in the drug discovery process. However, chemogenomics, which enables the system-wide analysis of the effects of different compounds via a description (or s...
We have developed a system for site-specific DNA integration in human cells, mediated by the adeno-associated virus (AAV) Rep proteins. In its normal lysogenic cycle, AAV integrates at a site on human chromosome 19 termed AAVS1. We describe a rapid PCR assay for the detection of integration events at AAVS1 in whole populations of cells. Using this...
Capsid-targeted viral inactivation is a novel protein-based strategy for the treatment of viral infections. Virus particles are inactivated by targeting toxic fusion proteins to virions, where they destroy viral components from within. We have fused Staphylococcus nuclease (SN) to the C-terminal end of Moloney murine leukemia virus Gag and demonstr...
The human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vpr and Vpx proteins are packaged into virions through virus type-specific interactions with the Gag polyprotein precursor. To examine whether HIV-1 Vpr (Vpr1) and HIV-2 Vpx (Vpx2) could be used to target foreign proteins to the HIV particle, their open reading frames were fused in frame wit...
Capsid-targeted viral inactivation is an antiviral strategy in which toxic fusion proteins are targeted to virions, where they inhibit viral multiplication by destroying viral components. These fusion proteins consist of a virion structural protein moiety and an enzymatic moiety such as a nuclease. Such fusion proteins can severely inhibit transpos...
The Saccharomyces cerevisiae genome contains four loci that encode histone proteins. Two of these loci, HTA1-HTB1 and HTA2-HTB2,
each encode histones H2A and H2B. The other two loci, HHT1-HHF1 and HHT2-HHF2, each encode histones H3 and H4. Because of
their redundancy, deletion of any one histone locus does not cause lethality. Previous experiments...
The Saccharomyces cerevisiae genome contains four loci that encode histone proteins. Two of these loci, HTA1-HTB1 and HTA2-HTB2, each encode histones H2A and H2B. The other two loci, HHT1-HHF1 and HHT2-HHF2, each encode histones H3 and H4. Because of their redundancy, deletion of any one histone locus does not cause lethality. Previous experiments...
Mutations in the SPT10 and SPT21 genes were originally isolated as suppressors of Ty and LTR (delta) insertion mutations in Saccharomyces cerevisiae, and the genes were shown to be required for normal transcription at a number of loci in yeast. Now we have cloned, sequenced, mapped and mutagenized SPT10 and SPT21. Since the spt10 mutation used to c...
A collection of yeast strains bearing single marked Ty1 insertions on chromosome III was generated. Over 100 such insertions were physically mapped by pulsed-field gel electrophoresis. These insertions are very nonrandomly distributed. Thirty-two such insertions were cloned by the inverted PCR technique, and the flanking DNA sequences were determin...
The 3' long terminal repeat (LTR) of yeast transposon Ty1 is not normally used as a promoter, although it contains sequences identical to those found in the 5' LTR, which does act as a promoter. We have isolated mutations that fall into two genes, SPT10 and SPT21, that allow the 3' LTRs of Ty1 elements inserted at various positions in the genome of...
Haploid yeast strains bearing approximately double the normal number of Ty1 elements have been constructed using marked GAL/Ty1 fusion plasmids. The strains maintain their high transposon copy number and overall genome structure in the absence of selection. The strains bearing extra Ty1 copies are surprisingly similar phenotypically to the parental...
Overexpression of dominant-negative mutants of various viral proteins can result in 'intracellular immunization'. Here we describe a new approach to interfering with viral replication in which a nuclease is fused to a capsid component so that the nuclease is encapsidated inside the virion where it can inactivate viral nucleic acid. We used Ty1, a y...
A large collection of Ty1 insertions in the URA3 and LYS2 loci was generated using a GAL1-Ty1 fusion to augment the transposition frequency. The sites of insertion of most of these Ty elements were sequenced. There appears to be a gradient of frequency of insertion from the 5' end (highest frequency) to the 3' end (lowest frequency) of both loci. I...
5-FOA is an extremely useful reagent for the selection of Ura- cells amid a population of Ura+ cells. The selection is effective in transformation and recombination studies where loss of URA3+ is desired. A new plasmid shuffling procedure based on the 5-FOAR selection permits the recovery of conditional lethal mutations in cloned genes that encode...
The gene encoding the histidine-tRNA synthetase (HTS1) has two in-frame translation start sites located 60 bp apart. One set of HTS1 transcripts (long) initiates upstream of both ATG codons, and the other set (short) initiates between the two ATG codons and therefore contains only the downstream ATG. A mutation that destroys the first AUG on the lo...
TheSaccharomyces cerevisiae genome contains fourloci thatencodehistone proteins. Twooftheseloci, HTAJ-HTBIandHTA2-HTB2, eachencodehistones H2AandH2B.Theothertwoloci, HHTI-HHFIand HHT2-HHF2, eachencode histones H3andH4.Because oftheir redundancy, deletion ofany one histone locus doesnotcauselethality. Previous experiments demonstrated thatmutations...