Pharmacogenetic allele nomenclature: International workgroup recommendations for test result reporting.

This article provides nomenclature recommendations developed by an international workgroup to increase transparency and standardization of pharmacogenetic (PGx) result reporting. Presently, sequence variants identified by PGx tests are described using different nomenclature systems. In addition, PGx analysis may detect different sets of variants for each gene, which can affect interpretation of results. This practice has caused confusion and may thereby impede the adoption of clinical PGx testing. Standardization is critical to move PGx forward.

A genome-wide analysis of biomineralization-related proteins in the sea urchin Strongylocentrotus purpuratus.

Biomineralization, the biologically controlled formation of mineral deposits, is of widespread importance in biology, medicine, and engineering. Mineralized structures are found in most metazoan phyla and often have supportive, protective, or feeding functions. Among deuterostomes, only echinoderms and vertebrates produce extensive biomineralized structures. Although skeletons appeared independently in these two groups, ancestors of the vertebrates and echinoderms may have utilized similar components of a shared genetic "toolkit" to carry out biomineralization. The present study had two goals. First, we sought to expand our understanding of the proteins involved in biomineralization in the sea urchin, a powerful model system for analyzing the basic cellular and molecular mechanisms that underlie this process. Second, we sought to shed light on the possible evolutionary relationships between biomineralization in echinoderms and vertebrates. We used several computational methods to survey the genome of the purple sea urchin Strongylocentrotus purpuratus for gene products involved in biomineralization. Our analysis has greatly expanded the collection of biomineralization-related proteins. We have found that these proteins are often members of small families encoded by genes that are clustered in the genome. Most of the proteins are sea urchin-specific; that is, they have no apparent homologues in other invertebrate deuterostomes or vertebrates. Similarly, many of the vertebrate proteins that mediate mineral deposition do not have counterparts in the S. purpuratus genome. Our findings therefore reveal substantial differences in the primary sequences of proteins that mediate biomineral formation in echinoderms and vertebrates, possibly reflecting loose constraints on the primary structures of the proteins involved. On the other hand, certain cellular and molecular processes associated with earlier events in skeletogenesis appear similar in echinoderms and vertebrates, leaving open the possibility of deeper evolutionary relationships.

A radiation hybrid map of mouse genes.

A comprehensive gene-based map of a genome is a powerful tool for genetic studies and is especially useful for the positional cloning and positional candidate approaches. The availability of gene maps for multiple organisms provides the foundation for detailed conserved-orthology maps showing the correspondence between conserved genomic segments. These maps make it possible to use cross-species information in gene hunts and shed light on the evolutionary forces that shape the genome. Here we report a radiation hybrid map of mouse genes, a combined project of the Whitehead Institute/Massachusetts Institute of Technology Center for Genome Research, the Medical Research Council UK Mouse Genome Centre, and the National Center for Biotechnology Information. The map contains 11,109 genes, screened against the T31 RH panel and positioned relative to a reference map containing 2,280 mouse genetic markers. It includes 3,658 genes homologous to the human genome sequence and provides a framework for overlaying the human genome sequence to the mouse and for sequencing the mouse genome.

RefSeq and LocusLink: NCBI gene-centered resources.

Thousands of genes have been painstakingly identified and characterized a few genes at a time. Many thousands more are being predicted by large scale cDNA and genomic sequencing projects, with levels of evidence ranging from supporting mRNA sequence and comparative genomics to computing ab initio models. This, coupled with the burgeoning scientific literature, makes it critical to have a comprehensive directory for genes and reference sequences for key genomes. The NCBI provides two resources, LocusLink and RefSeq, to meet these needs. LocusLink organizes information around genes to generate a central hub for accessing gene-specific information for fruit fly, human, mouse, rat and zebrafish. RefSeq provides reference sequence standards for genomes, transcripts and proteins; human, mouse and rat mRNA RefSeqs, and their corresponding proteins, are discussed here. Together, RefSeq and LocusLink provide a non-redundant view of genes and other loci to support research on genes and gene families, variation, gene expression and genome annotation. Additional information about LocusLink and RefSeq is available at http://www.ncbi.nlm.nih.gov/LocusLink/.

A fast and scalable radiation hybrid map construction and integration strategy.

This paper describes a fast and scalable strategy for constructing a radiation hybrid (RH) map from data on different RH panels. The maps on each panel are then integrated to produce a single RH map for the genome. Recurring problems in using maps from several sources are that the maps use different markers, the maps do not place the overlapping markers in same order, and the objective functions for map quality are incomparable. We use methods from combinatorial optimization to develop a strategy that addresses these issues. We show that by the standard objective functions of obligate chromosome breaks and maximum likelihood, software for the traveling salesman problem produces RH maps with better quality much more quickly than using software specifically tailored for RH mapping. We use known algorithms for the longest common subsequence problem as part of our map integration strategy. We demonstrate our methods by reconstructing and integrating maps for markers typed on the Genebridge 4 (GB4) and the Stanford G3 panels publicly available from the RH database. We compare map quality of our integrated map with published maps for GB4 panel and G3 panel by considering whether markers occur in the same order on a map and in DNA sequence contigs submitted to GenBank. We find that all of the maps are inconsistent with the sequence data for at least 50% of the contigs, but our integrated maps are more consistent. The map integration strategy not only scales to multiple RH maps but also to any maps that have comparable criteria for measuring map quality. Our software improves on current technology for doing RH mapping in areas of computation time and algorithms for considering a large number of markers for mapping. The essential impediments to producing dense high-quality RH maps are data quality and panel size, not computation.

NCBI's LocusLink and RefSeq.

The NCBI has introduced two new web resources-LocusLink and RefSeq-that facilitate retrieval of gene-based information and provide reference sequence standards. These resources are designed to provide a non-redundant view of current knowledge about human genes, transcripts and proteins. Additional information about these resources is available on the LocusLink web site at http://www.ncbi.nlm.nih.gov/LocusLink/

NCBI genetic resources supporting immunogenetic research.

The NCBI creates and maintains a set of integrated bibliographic, sequence, map, structure and other database resources to promote the efficient retrieval of information and the discovery of novel relationships. The connections made between elements of these resources permit researchers to start a search from a wide spectrum of entry points. These multiple dimensions of data can be roughly categorized by primary content as text or bibliographic (PubMed, PubMedCentral, OMIM, LocusLink), sequence (GenBank, Reference Sequence Project (RefSeq), dbSNP, MMDB), protein structure (MMDB) or map position (MapView). They can also becategorized by level of expert curation, which may range from validation of submissions from external groups (GenBank, PubMed, PubMedCentral,), to automatic computation (HomoloGene, UniGene), and to highly reviewed and corrected (LocusLink, MMDB, OMIM, RefSeq). Searches can be made by words (in an article title, key words, sequence annotation, database value, author) by sequence (BLAST or e-PCR against multiple sequence databases), or by map coordinates. By computing or curating bi-directional links between related objects, NCBI can represent content on the genetics, molecular biology, and clinical considerations of interest to immunogeneticists. There is also an emerging resource developed by the NCBI in collaboration with the IHWG devoted to the presentation of MHC data (dbMHC). How dbMHC will augment existing resources at the NCBI is described.

The human proteinase-activated receptor-3 (PAR-3) gene. Identification within a Par gene cluster and characterization in vascular endothelial cells and platelets.

Proteolytically activated receptors (PARs) represent an emerging subset of seven transmembrane G protein-coupled receptors that mediate cell activation events by receptor cleavage at distinct scissile bonds located within receptor amino termini. Differential genomic blotting using a yeast artificial chromosome known to contain the PAR-1 and PAR-2 genes identified the PAR-3 gene within a PAR gene cluster spanning approximately 100 kilobases at 5q13. The PAR-3 gene is relatively small (approximately 12 kilobases); and, like the PAR-1 and PAR-2 genes, it displays a two-exon structure, with the majority of the coding sequence and the proteolytic cleavage site contained within the larger second exon. Sequence analysis of the 5'-flanking region demonstrates that the promoter is TATA-less, similar to that seen with PAR-1, with the identification of nucleic acid motifs potentially involved in transcriptional gene regulation, including AP-1, GATA, and octameric sequences. PAR-3 transcripts were apparent in human vascular endothelial cells, although at considerably lower levels than those of PAR-1 and not significantly modulated by the endothelial cell stimulus tumor necrosis factor-alpha. Likewise, although PAR-3 mRNA was evident in human platelets, receptor cell surface expression was modest (approximately 10%) compared with that of PAR-1. Thus, although PAR-3 is postulated to represent a second thrombin receptor, its modest endothelial cell and platelet expression suggest that PAR-3 activation by alpha-thrombin is less relevant for physiological responses in these mature cells. Rather, given its disparately greater expression in megakaryocytes (and megakaryocyte-like human erythroleukemia cells), a regulatory role in cellular development (by protease activation) could be postulated.

The human thrombin receptor and proteinase activated receptor-2 genes are tightly linked on chromosome 5q13.

The thrombin receptor (TR) and proteinase activated receptor-2 (PAR-2) may represent the prototypes of an emerging family of cell-surface receptors that effect cell activation events mediated by serine proteases generated during inflammatory, fibrinolytic or haemostatic-regulated pathways. To further characterize the molecular genetics of these receptors, we have refined the genetic and physical mapping of both PAR-2 and TR. Utilization of two distinct radiation hybrid mapping panels with different levels of resolution demonstrated that both genes are tightly linked to the microsatellite markers D5S424, D5S1977, D5S2529 and D5S2596 (in order of decreasing LOD scores, from 13.7 for D5S424 to 7.7 for D5S2596). Physical mapping using yeast artificial chromosomes (YACs) and inversion field gel electrophoresis demonstrated that they are maximally separate by 90 kb. If the association of TR and PAR-2 genes resulted from a relatively recent gene duplication event from a common ancestral gene, these observations provide a general framework for the identification of gene transcripts representing alternative proteolytically activated receptors which may be clustered within this region of the human genome. These observations are especially relevant given recent evidence that murine and human platelets express alternative signalling mechanisms or receptors for thrombin.

Regional assignment and tissue expression of twenty-three expressed sequence tags (ESTs) from human chromosome 5.

Regional localization and expression patterns are reported for 19 expressed sequence tags (ESTs) from human chromosome 5, two of which were derived from the same transcript. Two of the ESTs correspond to genes not previously characterized in humans: a stress-activated protein kinase and nicotinamide nucleotide transhydrogenase. Expression was determined by three methods: Northern blots, PCR from tissue-specific cDNA libraries, and sequence sampling from EST sequencing projects. Six of the ESTs show no expression, and EST01986 appears to be expressed predominantly in the brain by all methods tested.

Integration of transcript and genetic maps of chromosome 16 at near-1-Mb resolution: demonstration of a "hot spot" for recombination at 16p12.

A single mapping resource, a mouse/human somatic cell panel with average distance between breakpoints of 1.2 Mb and a potential resolution of 1 Mb, has been utilized to integrate the genetic map and a transcript map of human chromosome 16. This map includes 141 genetic markers and 200 genes and transcripts. The localization of four genes (CHEL3, TK2, TRG1, and MMP9) reported to map to chromosome 16 could not be confirmed, and for three of these localizations to other human chromosomes are reported. A correlation between genetic and physical distance over a region estimated to be 23 Mb on the short arm of chromosome 16 identified an interval demonstrating a greatly increased rate of recombination where, in females, 1 cM is equivalent to a physical distance of 100 kb.

Chromosome assignment of human brain expressed sequence tags (ESTs) by analyzing fluorescently labeled PCR products from hybrid cell panels.

Sixty-three human brain cDNA sequences were newly assigned to individual human chromosomes. Ten of these were subregionally localized, and one was also mapped in the mouse genome. Four previously reported assignments were refined. PCR primers were designed from expressed sequence tags (ESTs) and tested for specific amplification from human genomic DNA. DNA was then amplified, often in multiplexed PCR reactions, using DNA from somatic cell hybrid mapping panels as templates. The amplification products were identified using an automated fluorescence detection system. Chromosomal assignments were made by discordancy analysis. Thirteen newly localized cDNAs exhibited homology to previously reported sequences. EST01471 was shown to correspond to human microtubule-associated protein 1B (MAP1B), confirming the previous mapping of this gene to human chromosome 5. Other genes tentatively assigned to chromosomes based on these results were a component of the signal peptide receptor of the endoplasmic reticulum (EST00745) and a cyclic AMP-regulated phosphoprotein (EST01041) on chromosome 1, a protein phosphatase 2A 55-kDa regulatory subunit (EST01650) on chromosome 4, an NAD(P) transhydrogenase (EST01744) on chromosome 5, ribosomal proteins L1a or L1b (EST01627) and L18a (EST01583), a brain transcription factor (BF-1, EST00795) on chromosome 14, a milk fat globule membrane-related protein (EST01678) on chromosome 15, a putative peptide initiation factor (EST00675) on chromosome 17, thiosulfate sulfurtransferase (TST) on chromosome 22, and moesin (MSN) (EST00896) and a human equivalent of rat spot 14 (S14) (EST00887) on Xp11-->cen and Xpter-->p21.3, respectively.

Chromosomal assignment of 38 human brain expressed sequence tags (ESTs) by analyzing fluorescently labeled PCR products from hybrid cell panels.

We have localized 38 human brain cDNA sequences to individual human chromosomes. PCR primers were designed from expressed sequence tags and tested for specific amplification from human genomic DNA. The sizes of amplification products from DNA of somatic cell hybrid mapping panels were determined electrophoretically using an automated fluorescence detection system. Chromosomal assignments were made by discordancy analysis.