Precision medicine in the age of big data: The present and future role of large-scale unbiased sequencing in drug discovery and development.

High throughput molecular and functional profiling of patients is a key driver of precision medicine. DNA and RNA characterization has been enabled at unprecedented cost and scale through rapid, disruptive progress in sequencing technology, but challenges persist in data management and interpretation. We analyze the state-of-the-art of large-scale unbiased sequencing in drug discovery and development, including technology, application, ethical, regulatory, policy and commercial considerations, and discuss issues of LUS implementation in clinical and regulatory practice.

Identification of candidate genes for controlling development of the basilar pons by differential display PCR.

The basilar pons, a major hindbrain nucleus involved in sensory-motor integration, has become a model system for studying long-distance neuronal migration, axon-target recognition by collateral branching, and the formation of patterned axonal projections. To identify genes potentially involved in these developmental events, we have performed a differential display PCR screen comparing RNA isolated from the developing basilar pons with RNA obtained from developing cerebellum and olfactory bulb, as well as the mature basilar pons. Using 400 different combinations of primers, we screened more than 11,000 labeled DNA fragments and identified 201 that exhibited higher expression in the basilar pons than in the control tissues. From these, 138 distinct gene fragments were cloned. The differential expression of a large subset of these fragments was confirmed using RNase protection assays. In situ hybridization analysis revealed that the expression of many of these genes is limited to the basilar pons and only a few other brain regions, suggesting that they may play specific roles in pontine development.

Heparan sulfate proteoglycans as adhesive and anti-invasive molecules. Syndecans and glypican have distinct functions.

ARH-77 cells do not adhere to type I collagen and readily invade into collagen gels, but following expression of the transmembrane heparan sulfate proteoglycan syndecan-1, they bind collagen and fail to invade. We now show that cells transfected with syndecan-2 or syndecan-4 also bind collagen and are non-invasive. In contrast, cells transfected with the glycosylphosphatidylinositol-anchored proteoglycan glypican-1 do not bind to collagen and remain invasive, even though glypican- and syndecan-expressing cells have similar surface levels of heparan sulfate, and their proteoglycans have similar affinities for collagen. Analysis of cells expressing syndecan-1-glypican-1 chimeric proteoglycans reveals that inhibition of invasion requires the extracellular domain of syndecan but not its transmembrane or cytoplasmic domain. Surprisingly, cells bearing a chimera composed of the glypican extracellular domain fused to the syndecan transmembrane and cytoplasmic domains bind to collagen but remain invasive, implying that adhesion to collagen is not by itself sufficient to inhibit invasion. Apparently, the extracellular domain of syndecan-1, presumably by interacting with cell-surface signal transducing molecules, directly regulates complex cell behaviors such as motility and invasiveness. These results also show for the first time that syndecans and glypicans can have distinct functions, even when expressed by the same cell type.

Expression of the heparan sulfate proteoglycan glypican-1 in the developing rodent.

The glypicans are a family of glycosylphosphatidylinositol (GPI)-anchored proteoglycans that, by virtue of their cell-surface localization and possession of heparan sulfate chains, may regulate the responses of cells to numerous heparin-binding growth factors, cell adhesion molecules, and extracellular matrix components. Mutations in one glypican cause a syndrome of human birth defects, suggesting important roles for these proteoglycans in development. Glypican-1, the first-discovered member of this family, was originally found in cultured fibroblasts, and later shown to be a major proteoglycan of the mature and developing brain. Here we examine the pattern of glypican-1 mRNA and protein expression more widely in the developing rodent, concentrating on late embryonic and early postnatal stages. High levels of glypican-1 expression were found throughout the brain and skeletal system. In the brain, glypican-1 mRNA was widely, and sometimes only transiently, expressed by zones of neurons and neuroepithelia. Glypican-1 protein localized strongly to axons and, in the adult, to synaptic terminal fields as well. In the developing skeletal system, glypican-1 was found in the periosteum and bony trabeculae in a pattern consistent with expression by osteoblasts, as well as in the bone marrow. Glypican-1 was also observed in skeletal and smooth muscle, epidermis, and in the developing tubules and glomeruli of the kidney. Little or no expression was observed in the developing heart, lung, liver, dermis, or vascular endothelium at the stages examined. The tissue-, cell type-, and in some cases stage-specific expression of glypican-1 revealed in this study are likely to provide insight into the functions of this proteoglycan in development.

Cerebroglycan, a developmentally regulated cell-surface heparan sulfate proteoglycan, is expressed on developing axons and growth cones.

Cerebroglycan is a glycosylphosphatidylinositol-linked integral membrane heparan sulfate proteoglycan found exclusively in the developing nervous system. In the rodent, cerebroglycan mRNA first appears in regions containing newly generated neurons and typically disappears 1 to several days later (Stipp et al., 1994, J. Cell Biol. 124:149-160). To gain insight into the roles that cerebroglycan plays in the developing nervous system, monospecific antibodies were prepared and used to localize cerebroglycan protein. In the rat, cerebroglycan was prominantly expressed on axon tracts throughout the developing brain and spinal cord, where it was found at times when axons are actively growing, but generally not after axons have reached their targets. Cerebroglycan was also found on neuronal growth cones both in vivo and in vitro. Interestingly, cerebroglycan immunoreactivity was rarely seen in or around neuronal cell bodies. Indeed, by examining the hippocampus at a late stage in development-when most neurons no longer express cerebroglycan but newly generated granule neurons do-evidence was obtained that cerebroglycan is strongly polarized to the axonal, and excluded from the somatodendritic, compartment of neurons. The timing and pattern of cerebroglycan expression are consistent with a role for this cell-surface heparan sulfate proteoglycan in regulating the growth or guidance of axons.

Neuronal expression of glypican, a cell-surface glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan, in the adult rat nervous system.

Cell-surface proteoglycans have been implicated in cell responses to growth factors, extracellular matrix, and cell adhesion molecules. M12, one of the most abundant membrane-associated proteoglycans in the adult rat brain, is a approximately 65 kDa glycosylphosphatidylinositol-linked protein that bears heparan sulfate chains (Herndon and Lander, 1990). To assess its identity, M12 was purified and internal peptide sequences obtained. Comparison of the results with protein sequence predicted by a cDNA cloned from PC12 cells indicated that M12 is rat glypican, a proteoglycan first cloned from human fibroblasts. In addition, antibodies raised against a rat glypican fusion protein specifically detected the 65 kDa brain proteoglycan core protein, both by immunoprecipitation and by Western blotting. Northern blot analysis using a rat glypican probe also detected glypican message in the adult, as well as the developing rat brain. In situ hybridization with glypican RNA probes showed that glypican is expressed in a subset of structures in the adult rat nervous system. These include the hippocampus, dorsal thalamus, amygdala, cerebral cortex, piriform cortex, olfactory tubercle, several cranial nerve nuclei, the ventral horn of the spinal cord, and the dorsal root ganglia. Several other brain regions exhibited little or no hybridization over background. In most cases where glypican hybridization was observed, the signal could be localized specifically to the cell bodies of identifiable neurons, for example, spinal motoneurons, hippocampal pyramidal cells. In the cerebral cortex, glypican hybridization was found in layers 2/3, 5, and 6, but was missing from 1 and 4. The data suggest that glypican is expressed primarily by subpopulations of projection neurons in the adult rat nervous system.

Cerebroglycan: an integral membrane heparan sulfate proteoglycan that is unique to the developing nervous system and expressed specifically during neuronal differentiation.

Heparan sulfate proteoglycans (HSPGs) are found on the surface of all adherent cells and participate in the binding of growth factors, extracellular matrix glycoproteins, cell adhesion molecules, and proteases and antiproteases. We report here the cloning and pattern of expression of cerebroglycan, a glycosylphosphatidylinositol (GPI)-anchored HSPG that is found in the developing rat brain (previously referred to as HSPG M13; Herndon, M. E., and A. D. Lander. 1990. Neuron. 4:949-961). The cerebroglycan core protein has a predicted molecular mass of 58.6 kD and five potential heparan sulfate attachment sites. Together with glypican (David, G., V. Lories, B. Decock, P. Marynen, J.-J. Cassiman, and H. Van den Berghe. 1990. J. Cell Biol. 111:3165-3176), it defines a family of integral membrane HSPGs characterized by GPI linkage and conserved structural motifs, including a pattern of 14 cysteine residues that is absolutely conserved. Unlike other known integral membrane HSPGs, including glypican and members of the syndecan family of transmembrane proteoglycans, cerebroglycan is expressed in only one tissue: the nervous system. In situ hybridization experiments at several developmental stages strongly suggest that cerebroglycan message is widely and transiently expressed by immature neurons, appearing around the time of final mitosis and disappearing after cell migration and axon outgrowth have been completed. These results suggest that cerebroglycan may fulfill a function related to the motile behaviors of developing neurons.

Wide distribution of homologs of Escherichia coli Kdp K+-ATPase among gram-negative bacteria.

We used Southern blotting to screen a variety of bacterial genes for homology to the kdp genes of Escherichia coli, genes that encode an ATP-driven K+ transport system. We found that most enterobacteria have sequences homologous to those of the three kdp structural genes and the kdpD regulatory gene. A number of distantly related species, including some cyanobacteria, have sequences homologous to those of the structural genes but not the regulatory gene. In all cases only a single region of homology was found. These results suggest that ATP-driven transport systems similar to the Kdp system in structure and regulation are found in many enteric organisms. In other gram-negative organisms, the ATPase is more divergent, retaining good homology at the DNA level only to the highly conserved phosphorylated subunit of the ATPase.