Discovery of novel plasma biomarkers for future incident venous thromboembolism by untargeted synchronous precursor selection mass spectrometry proteomics.

Essentials Discovery of predictive biomarkers of venous thromboembolism (VTE) may aid risk stratification. A case-control study where plasma was sampled before the occurrence of VTE was established. We generated untargeted plasma proteomic profiles of 200 individuals by use of mass spectrometry. Assessment of the biomarker potential of 501 proteins yielded 46 biomarker candidates. ABSTRACT: Background Prophylactic anticoagulant treatment may substantially reduce the incidence of venous thromboembolism (VTE) but entails considerable risk of severe bleeding. Identification of individuals at high risk of VTE through the use of predictive biomarkers is desirable in order to achieve a favorable benefit-to-harm ratio. Objective We aimed to identify predictive protein biomarker candidates of VTE. Methods We performed a case-control study of 200 individuals that participated in the Tromsø Study, a population-based cohort, where blood samples were collected before the VTE events occurred. Untargeted tandem mass tag-synchronous precursor selection-mass spectrometry (TMT-SPS-MS3)-based proteomic profiling was used to study the plasma proteomes of each individual. Results Of the 501 proteins detected in a sufficient number of samples to allow multivariate analysis, 46 proteins were associated with VTE case-control status with P-values below the 0.05 significance threshold. The strongest predictive biomarker candidates, assessed by statistical significance, were transthyretin, vitamin K-dependent protein Z and protein/nucleic acid deglycase DJ-1. Conclusions Our untargeted approach of plasma proteome profiling revealed novel predictive biomarker candidates of VTE and confirmed previously reported candidates, thereby providing conceptual support for the validity of the study. A larger nested case-control study will be conducted to validate our findings.

Joint effects of prothrombotic genotypes and body height on the risk of venous thromboembolism: the Tromsø study.

Essentials Body height and prothrombotic genotypes are associated with risk of venous thromboembolism (VTE). The joint effect of prothrombotic genotypes and tall stature on VTE risk is scarcely investigated. We investigated the joint effect of prothrombotic genotypes and tall stature on VTE risk. Prothrombotic genotypes did not yield excess risk of VTE in subjects with a tall stature. SUMMARY: Background Studies have reported synergistic effects of prothrombotic single-nucleotide polymorphisms (SNPs) and obesity on the risk of venous thromboembolism (VTE). Tall stature is associated with an increased VTE risk, but the joint effect of prothrombotic genotypes and tall stature on the VTE risk is unknown. Aims To investigate the joint effects of prothrombotic genotypes and tall stature on the VTE risk. Methods Cases with incident VTE (n = 676) and a randomly selected age-weighted subcohort (n = 1842) were sampled from the Tromsø study (cohort follow-up: 1994-2012). DNA was genotyped for rs6025 (factor V Leiden), rs1799963 (FII), rs8176719 (ABO blood group), rs2066865 (fibrinogen-γ), and rs2036914 (FIX). Age-adjusted and sex-adjusted hazard ratios (HRs) of VTE were calculated by categories of risk alleles (de Haan 5-SNP score: 0-1, 2-3, and ≥ 4) and body height (< 40th, 40th-80th and > 80th percentiles). Results The VTE risk increased by increasing category of body height, and subjects with height ≥ 178 cm had a two-fold higher VTE risk (HR 2.03; 95% confidence interval [CI] 1.51-2.73) than those with height ≤ 165 cm. The VTE risk also increased across categories of risk alleles. However, the combination of a tall stature and risk alleles, either individual SNPs or risk score, did not result in an excess VTE risk. Subjects with four or more risk alleles and height ≥ 178 cm had a two-fold (HR 2.08; 95% CI 1.24-3.52) higher VTE risk than subjects ≤ 165 cm with no risk allele or one risk allele. Conclusions In contrast to obesity, the presence of prothrombotic genotypes did not result in an excess VTE risk in subjects with a tall stature.

Genetic and epigenetic profiling of CLL disease progression reveals limited somatic evolution and suggests a relationship to memory-cell development.

We examined genetic and epigenetic changes that occur during disease progression from indolent to aggressive forms of chronic lymphocytic leukemia (CLL) using serial samples from 27 patients. Analysis of DNA mutations grouped the leukemia cases into three categories: evolving (26%), expanding (26%) and static (47%). Thus, approximately three-quarters of the CLL cases had little to no genetic subclonal evolution. However, we identified significant recurrent DNA methylation changes during progression at 4752 CpGs enriched for regions near Polycomb 2 repressive complex (PRC2) targets. Progression-associated CpGs near the PRC2 targets undergo methylation changes in the same direction during disease progression as during normal development from naive to memory B cells. Our study shows that CLL progression does not typically occur via subclonal evolution, but that certain CpG sites undergo recurrent methylation changes. Our results suggest CLL progression may involve developmental processes shared in common with the generation of normal memory B cells.

Blocks of limited haplotype diversity revealed by high-resolution scanning of human chromosome 21.

Global patterns of human DNA sequence variation (haplotypes) defined by common single nucleotide polymorphisms (SNPs) have important implications for identifying disease associations and human traits. We have used high-density oligonucleotide arrays, in combination with somatic cell genetics, to identify a large fraction of all common human chromosome 21 SNPs and to directly observe the haplotype structure defined by these SNPs. This structure reveals blocks of limited haplotype diversity in which more than 80% of a global human sample can typically be characterized by only three common haplotypes.

Evolutionarily conserved sequences on human chromosome 21.

Comparison of human sequences with the DNA of other mammals is an excellent means of identifying functional elements in the human genome. Here we describe the utility of high-density oligonucleotide arrays as a rapid approach for comparing human sequences with the DNA of multiple species whose sequences are not presently available. High-density arrays representing approximately 22.5 Mb of nonrepetitive human chromosome 21 sequence were synthesized and then hybridized with mouse and dog DNA to identify sequences conserved between humans and mice (human-mouse elements) and between humans and dogs (human-dog elements). Our data show that sequence comparison of multiple species provides a powerful empiric method for identifying actively conserved elements in the human genome. A large fraction of these evolutionarily conserved elements are present in regions on chromosome 21 that do not encode known genes.

Active conservation of noncoding sequences revealed by three-way species comparisons.

Human and mouse genomic sequence comparisons are being increasingly used to search for evolutionarily conserved gene regulatory elements. Large-scale human-mouse DNA comparison studies have discovered numerous conserved noncoding sequences of which only a fraction has been functionally investigated A question therefore remains as to whether most of these noncoding sequences are conserved because of functional constraints or are the result of a lack of divergence time.

PipMaker--a web server for aligning two genomic DNA sequences.

PipMaker (http://bio.cse.psu.edu) is a World-Wide Web site for comparing two long DNA sequences to identify conserved segments and for producing informative, high-resolution displays of the resulting alignments. One display is a percent identity plot (pip), which shows both the position in one sequence and the degree of similarity for each aligning segment between the two sequences in a compact and easily understandable form. Positions along the horizontal axis can be labeled with features such as exons of genes and repetitive elements, and colors can be used to clarify and enhance the display. The web site also provides a plot of the locations of those segments in both species (similar to a dot plot). PipMaker is appropriate for comparing genomic sequences from any two related species, although the types of information that can be inferred (e.g., protein-coding regions and cis-regulatory elements) depend on the level of conservation and the time and divergence rate since the separation of the species. Gene regulatory elements are often detectable as similar, noncoding sequences in species that diverged as much as 100-300 million years ago, such as humans and mice, Caenorhabditis elegans and C. briggsae, or Escherichia coli and Salmonella spp. PipMaker supports analysis of unfinished or "working draft" sequences by permitting one of the two sequences to be in unoriented and unordered contigs.

Faithful expression of the human 5q31 cytokine cluster in transgenic mice.

Interleukins -4, -5, and -13, cardinal cytokines produced by Th2 cells, are coordinately expressed and clustered in 150-kb syntenic regions on mouse chromosome 11 and human chromosome 5q31. We analyzed two sets of human yeast artificial chromosome transgenic mice that contained the 5q31 cytokines to assess whether conserved sequences required for their coordinate and cell-specific regulation are contained within the cytokine cluster itself. Human IL-4, IL-13, and IL-5 were expressed under Th2, but not Th1, conditions in vitro. Each of these cytokines was produced during infection with Nippostrongylus brasiliensis, a Th2-inducing stimulus, and human IL-4 was generated after activation of NK T cells in vivo. Consistently fewer cells produced the endogenous mouse cytokines in transgenic than in control mice, suggesting competition for stable expression between the mouse and human genes. These data imply the existence of both conserved trans-activating factors and cis-regulatory elements that underlie the coordinate expression and lineage specificity of the type 2 cytokine genes in lymphocytes.

Genomic interval engineering of mice identifies a novel modulator of triglyceride production.

To accelerate the biological annotation of novel genes discovered in sequenced regions of mammalian genomes, we are creating large deletions in the mouse genome targeted to include clusters of such genes. Here we describe the targeted deletion of a 450-kb region on mouse chromosome 11, which, based on computational analysis of the deleted murine sequences and human 5q orthologous sequences, codes for nine putative genes. Mice homozygous for the deletion had a variety of abnormalities, including severe hypertriglyceridemia, hepatic and cardiac enlargement, growth retardation, and premature mortality. Analysis of triglyceride metabolism in these animals demonstrated a several-fold increase in hepatic very-low density lipoprotein triglyceride secretion, the most prevalent mechanism responsible for hypertriglyceridemia in humans. A series of mouse BAC and human YAC transgenes covering different intervals of the 450-kb deleted region were assessed for their ability to complement the deletion induced abnormalities. These studies revealed that OCTN2, a gene recently shown to play a role in carnitine transport, was able to correct the triglyceride abnormalities. The discovery of this previously unappreciated relationship between OCTN2, carnitine, and hepatic triglyceride production is of particular importance because of the clinical consequence of hypertriglyceridemia and the paucity of genes known to modulate triglyceride secretion.

VISTA : visualizing global DNA sequence alignments of arbitrary length.

SUMMARY: VISTA is a program for visualizing global DNA sequence alignments of arbitrary length. It has a clean output, allowing for easy identification of similarity, and is easily configurable, enabling the visualization of alignments of various lengths at different levels of resolution. It is currently available on the web, thus allowing for easy access by all researchers. AVAILABILITY: VISTA server is available on the web at http://www-gsd.lbl.gov/vista. The source code is available upon request. CONTACT: vista@lbl.gov

Identification of a coordinate regulator of interleukins 4, 13, and 5 by cross-species sequence comparisons.

Long-range regulatory elements are difficult to discover experimentally; however, they tend to be conserved among mammals, suggesting that cross-species sequence comparisons should identify them. To search for regulatory sequences, we examined about 1 megabase of orthologous human and mouse sequences for conserved noncoding elements with greater than or equal to 70% identity over at least 100 base pairs. Ninety noncoding sequences meeting these criteria were discovered, and the analysis of 15 of these elements found that about 70% were conserved across mammals. Characterization of the largest element in yeast artificial chromosome transgenic mice revealed it to be a coordinate regulator of three genes, interleukin-4, interleukin-13, and interleukin-5, spread over 120 kilobases.

Photoreceptor localization of the KIF3A and KIF3B subunits of the heterotrimeric microtubule motor kinesin II in vertebrate retina.

The heterotrimeric microtubule motor kinesin II has been shown to be required for morphogenesis and maintenance of both motile flagella and immotile sensory cilia. Recently, we showed that the KIF3A subunit of kinesin II is concentrated in the inner segment and connecting cilium of fish photoreceptors. Here we report the gene structure of human KIF3A (HsKIF3A) and describe its localization in human and monkey retina. We also describe the localization of both KIF3A and KIF3B kinesin II subunits in Xenopus retina. Using a portion of HsKIF3A we had amplified from adult human retinal cDNA, we found by a GenBank database search that an identical sequence had already been obtained by the Human Genome Center at Lawrence Berkeley National Laboratories in a direct sequencing analysis of 680 kb of human chromosome 5q31. By comparing the genomic sequence of HsKIF3A to the open reading frame (ORF) of the highly homologous mouse Kif3A, we determined that the HsKIF3A gene has 17 exons and an ORF of approximately 2.1 kb, predicting a protein of 80.3 kDa. Antibodies against sea urchin KRP85, a KIF3A homologue, bound to a single band of approximately 85 kDa in immunoblots of total retina protein from human, monkey and Xenopus. In these same samples, a single band of approximately 95 kDa is recognized by antibodies against Xklp3, a Xenopus KIF3B homologue. In sections of Xenopus retina, both antibodies strongly labelled photoreceptor inner segments and the outer limiting membrane. Both antibodies also labelled photoreceptor axonemes. The axonemal localization of kinesin II subunits suggests that kinesin II may play a role in transport of materials from the photoreceptor cell body to the outer segment.

Functional screening of an asthma QTL in YAC transgenic mice.

Many quantitative trait loci (QTLs) contributing to genetically complex conditions have been discovered, but few causative genes have been identified. This is mainly due to the large size of QTLs and the subtle connection between genotype and quantitative phenotype associated with these conditions. Transgenic mice have been successfully used to analyse well-characterized genes suspected of contributing to quantitative traits. Although this approach is powerful for examining one gene at a time, it can be impractical for surveying the large genomic intervals containing many genes that are typically associated with QTLs. To screen for genes contributing to an asthma QTL mapped to human chromosome 5q3 (refs 6,7), we characterized a panel of large-insert 5q31 transgenics based on studies demonstrating that altering gene dosage frequently affects quantitative phenotypes normally influenced by that gene. This panel of human YAC transgenics, propagating a 1-Mb interval of chromosome 5q31 containing 6 cytokine genes and 17 partially characterized genes, was screened for quantitative changes in several asthma-associated phenotypes. Multiple independent transgenic lines with altered IgE response to antigen treatment shared a 180-kb region containing 5 genes, including those encoding human interleukin 4 (IL4) and interleukin 13 (IL13 ), which induce IgE class switching in B cells. Further analysis of these mice and mice transgenic for mouse Il4 and Il13 demonstrated that moderate changes in Il4 and Il13 expression affect asthma-associated phenotypes in vivo. This functional screen of large-insert transgenics enabled us to identify genes that influence the QTL phenotype in vivo.

Computational and biological analysis of 680 kb of DNA sequence from the human 5q31 cytokine gene cluster region.

With the human genome project advancing into what will be a 7- to 10-year DNA sequencing phase, we are presented with the challenge of developing strategies to convert genomic sequence data, as they become available, into biologically meaningful information. We have analyzed 680 kb of noncontiguous DNA sequence from a 1-Mb region of human chromosome 5q31, coupling computational analysis with gene expression studies of tissues isolated from humans as well as from mice containing human YAC transgenes. This genomic interval has been noted previously for containing the cytokine gene cluster and a quantitative trait locus associated with inflammatory diseases. Our analysis identified and verified expression of 16 new genes, as well as 7 previously known genes. Of the total of 23 genes in this region, 78% had similarity matches to sequences in protein databases and 83% had exact expressed sequence tag (EST) database matches. Comparative mapping studies of eight of the new human genes discovered in the 5q31 region revealed that all are located in the syntenic region of mouse chromosome 11q. Our analysis demonstrates an approach for examining human sequence as it is made available from large sequencing programs and has resulted in the discovery of several biomedically important genes, including a cyclin, a transcription factor that is homologous to an oncogene, a protein involved in DNA repair, and several new members of a family of transporter proteins.

The apolipoprotein(a) gene is regulated by sex hormones and acute-phase inducers in YAC transgenic mice.

High plasma concentrations of apolipoprotein (a) (apo(a)) have been implicated as a major independent risk factor for atherosclerosis in humans. Apo(a) is a large, evolutionarily new gene (present primarily in primates) for which considerable controversy exists concerning the factors that regulate its expression. To investigate the in vivo regulation of apo(a), we have created several lines of YAC transgenic mice containing a 110-kb human apo(a) gene surrounded by greater than 60 kb of 5' and 3' flanking DNA. Studies in humans have suggested that acute-phase inducers increase and sex steroids decrease apo(a) concentrations, but these results are controversial. Analysis of the YAC transgenic mice conclusively supports the hypothesized role of sex steroids and refutes the suggested role of acute-phase inducers in regulating the apo(a) gene.

A radiation hybrid map of the region on human chromosome 22 containing the neurofibromatosis type 2 locus.

We describe a high-resolution radiation hybrid map of the region on human chromosome 22 containing the neurofibromatosis type 2 (NF2) gene. Eighty-five hamster-human somatic cell hybrids generated by X-irradiation and cell fusion were used to generate the radiation hybrid map. The presence or absence of 18 human chromosome 22-specific markers was determined in each hybrid by using Southern blot hybridization. Sixteen of the 18 markers were distinguishable by X-ray breakage in the radiation hybrids. Analysis of these data using two different mathematical models and two different statistical methods resulted in a single framework map consisting of 8 markers ordered with odds greater than 1000:1. The remaining nonframework markers were all localized to regions consisting of two adjoining intervals on the framework map with odds greater than 1000:1. Based on the RH map, the NF2 region of chromosome 22, defined by the flanking markers D22S1 and D22S28, is estimated to span a physical distance of approximately 6 Mb and is the most likely location for 9 of the 18 markers studied: D22S33, D22S41, D22S42, D22S46, D22S56, LIF, D22S37, D22S44, and D22S15.

Analysis of chromosome 22 deletions in neurofibromatosis type 2-related tumors.

The neurofibromatosis type 2 (NF2) gene has been hypothesized to be a recessive tumor suppressor, with mutations at the same locus on chromosome 22 that lead to NF2 also leading to sporadic tumors of the types seen in NF2. Flanking markers for this gene have previously been defined as D22S1 centromeric and D22S28 telomeric. Identification of subregions of this interval that are consistently rearranged in the NF2-related tumors would aid in better defining the disease locus. To this end, we have compared tumor and constitutional DNAs, isolated from 39 unrelated patients with sporadic and NF2-associated acoustic neuromas, meningiomas, schwannomas, and ependymomas, at eight polymorphic loci on chromosome 22. Two of the tumors studied revealed loss-of-heterozygosity patterns, which is consistent with the presence of chromosome 22 terminal deletions. By using additional polymorphic markers, the terminal deletion breakpoint found in one of the tumors, an acoustic neuroma from an NF2 patient, was mapped within the previously defined NF2 region. The breakpoint occurred between the haplotyped markers D22S41/D22S46 and D22S56. This finding redefines the proximal flanking marker and localizes the NF2 gene between markers D22S41/D22S46 and D22S28. In addition, we identified a sporadic acoustic neuroma that reveals a loss-of-heterozygosity pattern consistent with mitotic recombination or deletion and reduplication, which are mechanisms not previously seen in studies of these tumors. This finding, while inconsistent with models of tumorigenesis that invoke single deletions and their gene-dosage effects, lends further support to the recessive tumor-suppressor model.

Isolation and characterization of a laminin-binding protein from rat and chick muscle.

A major laminin-binding protein (LBP), distinct from previously described LBPs, has been isolated from chick and rat skeletal muscle (Mr 56,000 and 66,000, respectively). The purified LBPs from the two species were shown to be related antigenically and to have similar NH2-terminal amino acid sequences and total amino acid compositions. Protein blots using laminin and laminin fragments provided evidence that this LBP interacts with the major heparin-binding domain, E3, of laminin. Studies on the association of this LBP with muscle membrane fractions and reconstituted lipid vesicles indicate that this protein can interact with lipid bilayers and has properties of a peripheral, not an integral membrane protein. These properties are consistent with its amino acid sequence, determined from cDNAs (Clegg et al., 1988). Examination by light and electron microscopy of the LBP antigen distribution in skeletal muscle indicated that the protein is localized primarily extracellularly, near the extracellular matrix and myotube plasmalemma. While a form of this LBP has been identified in heart muscle, it is present at low or undetectable levels in other tissues examined by immunocytochemistry indicating that it is probably a muscle-specific protein. As this protein is localized extracellularly and can bind to both membranes and laminin, it may mediate myotube interactions with the extracellular matrix.