Showing papers by "Broad Institute published in 2004"

EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy.

Abstract: Receptor tyrosine kinase genes were sequenced in nonsmall cell lung cancer (NSCLC) and matched normal tissue. Somatic mutations of the epidermal growth factor receptor gene EGFR were found in 15 of 58 unselected tumors from Japan and 1 of 61 from the United States. Treatment with the EGFR kinase inhibitor gefitinib (Iressa) causes tumor regression in some patients with NSCLC, more frequently in Japan. EGFR mutations were found in additional lung cancer samples from U.S. patients who responded to gefitinib therapy and in a lung adenocarcinoma cell line that was hypersensitive to growth inhibition by gefitinib, but not in gefitinibinsensitive tumors or cell lines. These results suggest that EGFR mutations may predict sensitivity to gefitinib. Protein kinase activation by somatic mutation or

Large-Scale Copy Number Polymorphism in the Human Genome

Abstract: The extent to which large duplications and deletions contribute to human genetic variation and diversity is unknown. Here, we show that large-scale copy number polymorphisms (CNPs) (about 100 kilobases and greater) contribute substantially to genomic variation between normal humans. Representational oligonucleotide microarray analysis of 20 individuals revealed a total of 221 copy number differences representing 76 unique CNPs. On average, individuals differed by 11 CNPs, and the average length of a CNP interval was 465 kilobases. We observed copy number variation of 70 different genes within CNP intervals, including genes involved in neurological function, regulation of cell growth, regulation of metabolism, and several genes known to be associated with disease.

Transcriptional regulatory code of a eukaryotic genome

Abstract: DNA-binding transcriptional regulators interpret the genome's regulatory code by binding to specific sequences to induce or repress gene expression. Comparative genomics has recently been used to identify potential cis-regulatory sequences within the yeast genome on the basis of phylogenetic conservation, but this information alone does not reveal if or when transcriptional regulators occupy these binding sites. We have constructed an initial map of yeast's transcriptional regulatory code by identifying the sequence elements that are bound by regulators under various conditions and that are conserved among Saccharomyces species. The organization of regulatory elements in promoters and the environment-dependent use of these elements by regulators are discussed. We find that environment-specific use of regulatory elements predicts mechanistic models for the function of a large population of yeast's transcriptional regulators.

The ENCODE (ENCyclopedia of DNA elements) Project

Abstract: The ENCyclopedia Of DNA Elements (ENCODE) Project aims to identify all functional elements in the human genome sequence. The pilot phase of the Project is focused on a specified 30 megabases (∼1%) of the human genome sequence and is organized as an international consortium of computational and laboratory-based scientists working to develop and apply high-throughput approaches for detecting all sequence elements that confer biological function. The results of this pilot phase will guide future efforts to analyze the entire human genome.

Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype

Abstract: Tetraodon nigroviridis is a freshwater puffer fish with the smallest known vertebrate genome. Here, we report a draft genome sequence with long-range linkage and substantial anchoring to the 21 Tetraodon chromosomes. Genome analysis provides a greatly improved fish gene catalogue, including identifying key genes previously thought to be absent in fish. Comparison with other vertebrates and a urochordate indicates that fish proteins have diverged markedly faster than their mammalian homologues. Comparison with the human genome suggests ∼900 previously unannotated human genes. Analysis of the Tetraodon and human genomes shows that whole-genome duplication occurred in the teleost fish lineage, subsequent to its divergence from mammals. The analysis also makes it possible to infer the basic structure of the ancestral bony vertebrate genome, which was composed of 12 chromosomes, and to reconstruct much of the evolutionary history of ancient and recent chromosome rearrangements leading to the modern human karyotype.

mTOR inhibition reverses Akt-dependent prostate intraepithelial neoplasia through regulation of apoptotic and HIF-1-dependent pathways

Abstract: Loss of PTEN function leads to activation of phosphoinositide 3-kinase (PI3K) signaling and Akt. Clinical trials are now testing whether mammalian target of rapamycin (mTOR) inhibition is useful in treating PTEN-null cancers. Here, we report that mTOR inhibition induced apoptosis of epithelial cells and the complete reversal of a neoplastic phenotype in the prostate of mice expressing human AKT1 in the ventral prostate. Induction of cell death required the mitochondrial pathway, as prostate-specific coexpression of BCL2 blocked apoptosis. Thus, there is an mTOR-dependent survival signal required downstream of Akt. Bcl2 expression, however, only partially restored intraluminal cell growth in the setting of mTOR inhibition. Expression profiling showed that Hif-1 alpha targets, including genes encoding most glycolytic enzymes, constituted the dominant transcriptional response to AKT activation and mTOR inhibition. These data suggest that the expansion of AKT-driven prostate epithelial cells requires mTOR-dependent survival signaling and activation of HIF-1 alpha, and that clinical resistance to mTOR inhibitors may emerge through BCL2 expression and/or upregulation of HIF-1 alpha activity.

Assessing the impact of population stratification on genetic association studies.

Abstract: Population stratification refers to differences in allele frequencies between cases and controls due to systematic differences in ancestry rather than association of genes with disease. It has been proposed that false positive associations due to stratification can be controlled by genotyping a few dozen unlinked genetic markers. To assess stratification empirically, we analyzed data from 11 case-control and case-cohort association studies. We did not detect statistically significant evidence for stratification but did observe that assessments based on a few dozen markers lack power to rule out moderate levels of stratification that could cause false positive associations in studies designed to detect modest genetic risk factors. After increasing the number of markers and samples in a case-cohort study (the design most immune to stratification), we found that stratification was in fact present. Our results suggest that modest amounts of stratification can exist even in well designed studies.

Activating Mutations of the Noonan Syndrome-Associated SHP2/PTPN11 Gene in Human Solid Tumors and Adult Acute Myelogenous Leukemia

Abstract: The SH2 domain-containing protein-tyrosine phosphatase PTPN11 (Shp2) is required for normal development and is an essential component of signaling pathways initiated by growth factors, cytokines, and extracellular matrix. In many of these pathways, Shp2 acts upstream of Ras. About 50% of patients with Noonan syndrome have germ-line PTPN11 gain of function mutations. Associations between Noonan syndrome and an increased risk of some malignancies, notably leukemia and neuroblastoma, have been reported, and recent data indicate that somatic PTPN11 mutations occur in children with sporadic juvenile myelomonocytic leukemia, myelodysplasic syndrome, B-cell acute lymphoblastic leukemia, and acute myelogenous leukemia (AML). Juvenile myelomonocytic leukemia patients without PTPN11 mutations have either homozygotic NF-1 deletion or activating RAS mutations. Given the role of Shp2 in Ras activation and the frequent mutation of RAS in human tumors, these data raise the possibility that PTPN11 mutations play a broader role in cancer. We asked whether PTPN11 mutations occur in other malignancies in which activating RAS mutations occur at low but significant frequency. Sequencing of PTPN11 from 13 different human neoplasms including breast, lung, gastric, and neuroblastoma tumors and adult AML and acute lymphoblastic leukemia revealed 11 missense mutations. Five are known mutations predicted to result in an activated form of Shp2, whereas six are new mutations. Biochemical analysis confirmed that several of the new mutations result in increased Shp2 activity. Our data demonstrate that mutations in PTPN11 occur at low frequency in several human cancers, especially neuroblastoma and AML, and suggest that Shp2 may be a novel target for antineoplastic therapy.

Methods for high-density admixture mapping of disease genes.

Abstract: Admixture mapping (also known as “mapping by admixture linkage disequilibrium,” or MALD) has been proposed as an efficient approach to localizing disease-causing variants that differ in frequency (because of either drift or selection) between two historically separated populations. Near a disease gene, patient populations descended from the recent mixing of two or more ethnic groups should have an increased probability of inheriting the alleles derived from the ethnic group that carries more disease-susceptibility alleles. The central attraction of admixture mapping is that, since gene flow has occurred recently in modern populations (e.g., in African and Hispanic Americans in the past 20 generations), it is expected that admixture-generated linkage disequilibrium should extend for many centimorgans. High-resolution marker sets are now becoming available to test this approach, but progress will require (a) computational methods to infer ancestral origin at each point in the genome and (b) empirical characterization of the general properties of linkage disequilibrium due to admixture. Here we describe statistical methods to estimate the ancestral origin of a locus on the basis of the composite genotypes of linked markers, and we show that this approach accurately estimates states of ancestral origin along the genome. We apply this approach to show that strong admixture linkage disequilibrium extends, on average, for 17 cM in African Americans. Finally, we present power calculations under varying models of disease risk, sample size, and proportions of ancestry. Studying ∼2,500 markers in ∼2,500 patients should provide power to detect many regions contributing to common disease. A particularly important result is that the power of an admixture mapping study to detect a locus will be nearly the same for a wide range of mixture scenarios: the mixture proportion should be 10%–90% from both ancestral populations.

A High-Density Admixture Map for Disease Gene Discovery in African Americans

Abstract: Admixture mapping (also known as “mapping by admixture linkage disequilibrium,” or MALD) provides a way of localizing genes that cause disease, in admixed ethnic groups such as African Americans, with ∼100 times fewer markers than are required for whole-genome haplotype scans. However, it has not been possible to perform powerful scans with admixture mapping because the method requires a dense map of validated markers known to have large frequency differences between Europeans and Africans. To create such a map, we screened through databases containing ∼450,000 single-nucleotide polymorphisms (SNPs) for which frequencies had been estimated in African and European population samples. We experimentally confirmed the frequencies of the most promising SNPs in a multiethnic panel of unrelated samples and identified 3,011 as a MALD map (1.2 cM average spacing).We estimate that this map is ∼70% informative in differentiating African versus European origins of chromosomal segments. This map provides a practical and powerful tool, which is freely available without restriction, for screening for disease genes in African American patient cohorts. The map is especially appropriate for those diseases that differ in incidence between the parental African and European populations.

Genetic Dissection of Complex Traits with Chromosome Substitution Strains of Mice

Abstract: Chromosome substitution strains (CSSs) have been proposed as a simple and powerful way to identify quantitative trait loci (QTLs) affecting developmental, physiological, and behavioral processes. Here, we report the construction of a complete CSS panel for a vertebrate species. The CSS panel consists of 22 mouse strains, each of which carries a single chromosome substituted from a donor strain (A/J) onto a common host background (C57BL/6J). A survey of 53 traits revealed evidence for 150 QTLs affecting serum levels of sterols and amino acids, diet-induced obesity, and anxiety. These results demonstrate that CSSs greatly facilitate the detection and identification of genes that control the wide diversity of naturally occurring phenotypic variation in the A/J and C57BL/6J inbred strains.

A Library of Spirooxindoles Based on a Stereoselective Three-Component Coupling Reaction

Abstract: A collection of structurally complex and chemically diverse small molecules is a useful tool to explore cell circuitry. In this article, we report the split-pool synthesis of more than 3000 spirooxindoles on high capacity macrobeads. The key reaction to assemble the spirooxindole core stereoselectively is a Lewis acid variant of the Williams' three-component coupling. After formation, the skeleton was elaborated using Sonogashira couplings, amide forming reactions, and N-acylations of gamma-lactams. The final library was analyzed by sampling individual macrobeads and by using binomial confidence limits. It was determined that at least 82% of the library compounds should have better than 80% purity. To demonstrate the utility of our discovery process, a high-throughput chemical genetic modifier screen was performed using stock solutions of the resultant products. A number of positives were identified as enhancers of the cellular actions of latrunculin B, an actin polymerization inhibitor. Through resynthesis, we confirmed one of the positives and demonstrated that, in yeast cells, it has an EC50 in the sub-micromolar range.

Genomewide linkage analysis of bipolar disorder by use of a high-density single-nucleotide-polymorphism (SNP) genotyping assay: a comparison with microsatellite marker assays and finding of significant linkage to chromosome 6q22.

Abstract: We performed a linkage analysis on 25 extended multiplex Portuguese families segregating for bipolar disorder, by use of a high-density single-nucleotide–polymorphism (SNP) genotyping assay, the GeneChip Human Mapping 10K Array (HMA10K). Of these families, 12 were used for a direct comparison of the HMA10K with the traditional 10-cM microsatellite marker set and the more dense 4-cM marker set. This comparative analysis indicated the presence of significant linkage peaks in the SNP assay in chromosomal regions characterized by poor coverage and low information content on the microsatellite assays. The HMA10K provided consistently high information and enhanced coverage throughout these regions. Across the entire genome, the HMA10K had an average information content of 0.842 with 0.21-Mb intermarker spacing. In the 12-family set, the HMA10K-based analysis detected two chromosomal regions with genomewide significant linkage on chromosomes 6q22 and 11p11; both regions had failed to meet this strict threshold with the microsatellite assays. The full 25-family collection further strengthened the findings on chromosome 6q22, achieving genomewide significance with a maximum nonparametric linkage (NPL) score of 4.20 and a maximum LOD score of 3.56 at position 125.8 Mb. In addition to this highly significant finding, several other regions of suggestive linkage have also been identified in the 25-family data set, including two regions on chromosome 2 (57 Mb, NPL = 2.98; 145 Mb, NPL = 3.09), as well as regions on chromosomes 4 (91 Mb, NPL = 2.97), 16 (20 Mb, NPL = 2.89), and 20 (60 Mb, NPL = 2.99). We conclude that at least some of the linkage peaks we have identified may have been largely undetected in previous whole-genome scans for bipolar disorder because of insufficient coverage or information content, particularly on chromosomes 6q22 and 11p11.

Globally distributed object identification for biological knowledgebases

Abstract: The World-Wide Web provides a globally distributed communication framework that is essential for almost all scientific collaboration, including bioinformatics. However, several limits and inadequacies have become apparent, one of which is the inability to programmatically identify locally named objects that may be widely distributed over the network. This shortcoming limits our ability to integrate multiple knowledgebases, each of which gives partial information of a shared domain, as is commonly seen in bioinformatics. The Life Science Identifier (LSID) and LSID Resolution System (LSRS) provide simple and elegant solutions to this problem, based on the extension of existing internet technologies. LSID and LSRS are consistent with next-generation semantic web and semantic grid approaches. This article describes the syntax, operations, infrastructure compatibility considerations, use cases and potential future applications of LSID and LSRS. We see the adoption of these methods as important steps toward simpler, more elegant and more reliable integration of the world’s biological knowledgebases, and as facilitating stronger global collaboration in biology.

Genome-wide scan in Portuguese Island families identifies 5q31-5q35 as a susceptibility locus for schizophrenia and psychosis.

Abstract: Schizophrenia is a common psychiatric disorder with a complex genetic etiology. To understand the genetic basis of this syndrome in Portuguese Island populations, we performed a genome-wide scan of 29 families with schizophrenia, which identified a single region on 5q31–5q35 with strong linkage (NPL=3.09, P=0.0012 at D5S820). Empirical simulations set a genome-wide threshold of NPL=3.10 for significant linkage. Additional support for this locus in schizophrenia comes from higher-density mapping and mapping of 11 additional families. The combined set of 40 families had a peak NPL=3.28 (P=0.00066) at markers D5S2112–D5S820. These data and previous linkage findings from other investigators provide strong and consistent evidence for this genomic region as a susceptibility locus for schizophrenia. Exploratory analyses of a novel phenotype, psychosis, in families with schizophrenia and bipolar disorder detected evidence for linkage to the same markers as found in schizophrenia (peak NPL=3.03, P=0.0012 at D5S820), suggesting that this locus may be responsible for the psychotic symptoms observed in both diseases.

Genome sequencing: Three's company

Abstract: Publication of the rat genome sequence will not only advance physiological studies in this paragon of laboratory animals, but also greatly enhance the power of comparative research into mammalian genomes.

Common variation in BRCA2 and breast cancer risk: a haplotype-based analysis in the Multiethnic Cohort

Abstract: It is well established that rare mutations in BRCA2 predispose to familial breast cancer, but whether common variants at this locus contribute more modest risk to sporadic breast cancer has not been thoroughly investigated. We performed a haplotype-based study of BRCA2 among women in the Multiethnic Cohort Study (MEC), genotyping 50 SNPs spanning 109.4 kb of the BRCA2 gene. Twenty-one haplotype-tagging SNPs (including seven missense SNPs) were selected to predict the common BRCA2 haplotypes and were genotyped in a breast cancer case-control study nested in the MEC (cases, n=1715; controls, n=2502). Compared to non-carriers, we observed nominally significant positive associations for homozygous carriers of specific haplotypes in blocks 2 (haplotype 2c: OR=1.50; 95% CI, 1.08-2.09) and 3 (haplotype 3d: OR=1.50; 95% CI, 1.01-2.24). These results could be explained on the basis of a single marker in intron 24 (SNP 42: rs206340) that was correlated with these haplotypes and the homozygous state was associated with a significantly increased risk of breast cancer (AA versus GG genotypes: OR=1.59, 95% CI, 1.18-2.16; nominal P=0.005). This association was modestly stronger among women with advanced disease (OR=2.00, 95% CI, 1.30-3.08; P=0.002). In this exploratory analysis, we found little indication that common variation in BRCA2 dramatically impacts sporadic breast cancer risk. However, a significant elevation in risk was observed among approximately 6% of women who carried a specific haplotype pattern and may harbor a susceptibility allele at the BRCA2 locus.

Chromosomes 6 and 13 harbor genes that regulate pubertal timing in mouse chromosome substitution strains.

Abstract: Variation in the onset of puberty among inbred strains of mice suggests that quantitative trait loci (QTLs) affect neurological and hormonal aspects of sexual maturation. Taking a novel approach toward identifying factors that regulate the hypothalamic-pituitary-gonadal (HPG) axis, we evaluated pubertal timing [as assessed by vaginal opening (VO)] in two inbred strains of mice, A/J and C57BL/6J (B6), and in a panel of chromosome substitution strains (CSSs) generated from A/J and B6 mice. In each CSS, a single chromosome from A/J has been substituted in a homozygous fashion for the corresponding chromosome in B6, partitioning the A/J genome into 22 strains with a common host (B6) background. VO occurred significantly earlier in A/J compared with B6 mice. Although the majority of the CSSs assessed had a timing of VO that was similar to the progenitor B6 strain, CSSs for chromosomes 6 and 13 each displayed significantly earlier time of VO than B6 mice. F1 (B6 x CSS) mice for chromosomes 6 and 13 displayed phenotypes that were intermediate between the CSS and B6 strains, suggesting that the trait was inherited in a codominant manner. These findings demonstrate that chromosomes 6 and 13 harbor QTLs that control the timing of VO. Identification of the responsible genes may reveal factors that regulate the maturation of the HPG axis and determine the timing of puberty.

Independence of ℓ of monodromy groups

Abstract: o-\ : Garith(?A,r/) ^ GL(C\f?). The philosophy of motives in algebraic geometry leads one to suspect that if our ^-compatible system L = {C\} is "motivic", then the collection {Garith(^A5 v)ia\} of ?-adic monodromy groups and their tautological representations should be "in dependent of ?" in a suitable sense. This philosophy can be made more precise in the form of the following conjecture, which we are going to address in this paper.

The Ensembl Computing Architecture

Abstract: Ensembl is a software project to automatically annotate large eukaryotic genomes and release them freely into the public domain. The project currently automatically annotates 10 complete genomes. This makes very large demands on compute resources, due to the vast number of sequence comparisons that need to be executed. To circumvent the financial outlay often associated with classical supercomputing environments, farms of multiple, lower-cost machines have now become the norm and have been deployed successfully with this project. The architecture and design of farms containing hundreds of compute nodes is complex and nontrivial to implement. This study will define and explain some of the essential elements to consider when designing such systems. Server architecture and network infrastructure are discussed with a particular emphasis on solutions that worked and those that did not (often with fairly spectacular consequences). The aim of the study is to give the reader, who may be implementing a large-scale biocompute project, an insight into some of the pitfalls that may be waiting ahead.

SNP Genotyping using Sequenom MassARRAY 7K Platform

Abstract: Many high-throughput single-nucleotide polymorphism (SNP) genotyping technologies are currently available. The method for SNP genotyping described in this unit is based on the commercially available Sequenom MassARRAY platform which offers several attractive features for users desiring an accurate SNP genotyping assay. The assay described is based on primer extension and offers two levels of specificity. First a locus-specific PCR reaction takes place, followed by a locus-specific primer extension reaction (homogeneous Mass Extend, or hME assay) in which an oligonucleotide primer anneals immediately upstream of the polymorphic site being genotyped. The extension of the primer is according to the sequence of the variant site and can be a single complementary base or a series of complementary bases. Through the use of MALDI-TOF mass spectrometry, the mass of the extended primer is determined. The preparation of probe and genomic DNA is also described in this unit. Keywords: genotyping; SNP; MassARRAY; high-throughput; PCR

Computational Problems in Perfect Phylogeny Haplotyping: Xor-Genotypes and Tag SNPs

Abstract: The perfect phylogeny model for haplotype evolution has been successfully applied to haplotype resolution from genotype data. In this study we explore the application of the perfect phylogeny model to other problems in the design and analysis of genetic studies. We consider a novel type of data, xor-genotypes, which distinguish heterozygote from homozygote sites but do not identify the homozygote alleles. We show how to resolve xor-genotypes under perfect phylogeny model, and study the degrees of freedom in such resolutions. Interestingly, given xor-genotypes that produce a single possible resolution, we show that the full genotype of at most three individuals suffice in order to determine all haplotypes across the phylogeny. Our experiments with xor-genotyping data indicate that the approach requires a number of individuals only slightly larger than full genotyping, at a potentially reduced typing cost.

Biological database design and implementation

Abstract: We present our experience of building biological databases. Such databases have most aspects in common with other complex databases in other fields. We do not believe that biological data are that different from complex data in other fields. Our experience has led us to emphasise simplicity and conservative technology choices when building these databases. This is a short paper of advice that we hope is useful to people designing their own biological database.