Mining the transcriptome for rare disease therapies: a comparison of the efficiencies of two data mining approaches and a targeted cell-based drug screen.

Most monogenic diseases can be viewed as conditions caused by dysregulated protein activity; therefore, drugs can be used to modulate gene expression, and thus protein level, possibly conferring clinical benefit. When considering repurposing drugs for loss of function diseases, there are three classes of genetic disease amenable to an increase of function; haploinsufficient dominant diseases, those secondary to hypomorphic recessive alleles, and conditions with rescuing paralogs. This therapeutic model then brings the questions: how frequently do such clinically useful drug-gene interactions occur and what is the most rapid and efficient route by which to identify them. Here we compare three approaches: (1) mining of pre-existing system-wide transcriptomal datasets such as Connectivity Map; (2) utilization of a proprietary causal reasoning engine knowledge base; and, (3) a targeted drug screen using clinically accepted agents tested against normal human fibroblasts. We have determined the validation rate of these approaches for 76 diseases (i.e., in vitro fibroblast mRNA increase); for the Connectivity Map, approximately 5% of tested putative drug-gene interactions validated, for causal reasoning engine knowledge base the rate was 10%, and for the targeted drug screen 9%. The degree of overlap between these methodologies was low suggesting they are complementary not redundant approaches to identify putative drug-gene interactions. Although the validation rate was low, a number of drug-gene interactions were successfully identified and are now being investigated for protein induction and in vivo effect. This analysis establishes potentially valuable therapeutic leads as well as useful benchmarks for the thousands of currently untreatable rare genetic conditions.

Activity and MeCP2-dependent regulation of nNOS levels in enteric neurons.

BACKGROUND: Rett syndrome (RTT) is a neurological disorder characterized by severe cognitive impairment, motor dyspraxia, and seizures. Rett syndrome arises predominantly from mutations in MECP2, the gene coding for methyl-CpG-binding protein 2 (MeCP2). MeCP2 is an important mediator of synaptic development and is essential in regulating homeostatic synaptic plasticity (HSP) in the brain. In addition to demonstrating central nervous system impairment, RTT patients also suffer from gastrointestinal (GI) dysmotility. We hypothesize that this is due to a similar impairment of plasticity-dependent synaptic function in the enteric nervous system (ENS). We recently reported that MeCP2 is expressed in the ENS, providing evidence that neuronal dysfunction may mediate the GI pathology. METHODS: Baseline measures of MeCP2-KO vs wild-type (WT) GI neuronal nitric oxide synthase (nNOS) were assessed in tissue samples and in vitro. Experiments were carried out to measure nNOS in baseline vs activated plasticity states in vitro. Functional in vivo studies were carried out to determine whether MeCP2-KO mice reproduced the RTT GI hypomotility. KEY RESULTS: Methyl-CpG-binding protein 2-KO mice reproduced the GI hypomotility seen in RTT. MeCP2-KO GI tissue demonstrated elevated nNOS levels. Cultured WT enteric neurons showed upregulation of nNOS following moderate, prolonged stimulation by hyperkalemia; neurons from MeCP2-KO mice failed to show this nNOS upregulation. CONCLUSIONS & INFERENCES: MeCP2 is required for proper GI motility and normal nNOS levels. Neuronal nitric oxide synthase imbalances could mediate the GI dysmotility seen in RTT. Disruption of MeCP2-dependent HSP may be the basis for aberrant nNOS levels and hence GI dysmotility in MeCP2-KO and RTT.

MeCP2 in the enteric nervous system.

BACKGROUND: Rett syndrome (RTT) is an intellectual deficit and movement disorder that develops during early childhood in girls. Affected children are normal until 6-18 months of age, after which symptoms begin to appear. Most cases of RTT are due to mutations in the MeCP2 gene leading to disruption of neuronal communication in the central nervous system. In addition, RTT patients show peripheral ailments such as gastrointestinal (GI), respiratory, and cardiac dysfunction. The etiology of intestinal dysfunction in RTT is not well-understood. Reports on the presence of MeCP2 in the peripheral nervous system are scant. As such we examined the levels of MeCP2 in human and murine GI tissue and assessed MeCP2 expression at various developmental stages. METHODS: Immunohistochemistry for MeCP2, HuC/D, juvenile beta tubulin, and GFAP was performed on human and murine intestine. Western blots of these same tissues were probed with MeCP2, vAChT, nNOS, and beta-actin antibodies. KEY RESULTS: MeCP2 is expressed throughout the GI tract. MeCP2 is expressed specifically in the enteric nervous system of the GI tract. MeCP2 is expressed in the GI tract throughout development with appearance beginning at or before E11.5 in the murine intestine. CONCLUSIONS & INFERENCES: The proof of MeCP2 expression in enteric neurons suggests that the GI dysmotility in Rett may arise from enteric network dysfunction secondary to MeCP2 mutation.

Striatal interneurons in dissociated cell culture.

In addition to the well-characterized direct and indirect projection neurons there are four major interneuron types in the striatum. Three contain GABA and either parvalbumin, calretinin or NOS/NPY/somatostatin. The fourth is cholinergic. It might be assumed that dissociated cell cultures of striatum (typically from embryonic day E18.5 in rat and E14.5 for mouse) contain each of these neuronal types. However, in dissociated rat striatal (caudate/putamen, CPu) cultures arguably the most important interneuron, the giant aspiny cholinergic neuron, is not present. When dissociated striatal neurons from E14.5 Sprague-Dawley rats were mixed with those from E18.5 rats, combined cultures from these two gestational periods yielded surviving cholinergic interneurons and representative populations of the other interneuron types at 5 weeks in vitro. Neurons from E12.5 CD-1 mice were combined with CPu neurons from E14.5 mice and the characteristics of striatal interneurons after 5 weeks in vitro were determined. All four major classes of interneurons were identified in these cultures as well as rare tyrosine hydroxylase positive interneurons. However, E14.5 mouse CPu cultures contained relatively few cholinergic interneurons rather than the nearly total absence seen in the rat. A later dissection day (E16.5) was required to obtain mouse CPu cultures totally lacking the cholinergic interneuron. We show that these cultures generated from two gestational age cells have much more nearly normal proportions of interneurons than the more common organotypic cultures of striatum. Interneurons are generated from both ages of embryos except for the cholinergic interneurons that originate from the medial ganglionic eminence of younger embryos. Study of these cultures should more accurately reflect neuronal processing as it occurs in the striatum in vivo. Furthermore, these results reveal a procedure for parallel culture of striatum and cholinergic depleted striatum that can be used to examine the function of the cholinergic interneuron in striatal networks.

Advances in EPA's indirect exposure modeling methodology.

The Environmental Protection Agency's National Center for Environmental Assessment is currently revising and updating its approach to modeling the health risks from indirect exposure to combustor emissions. The updated method is intended to provide the necessary tools for estimating these health risks and to provide the necessary algorithms for the calculation of contaminant concentrations in water bodies, soil, and the terrestrial and aquatic food chains resulting from the deposition and transfer of atmospheric pollutants. Significant additions to the approach include the expansion of the application of exposure methods at a site, the description of procedures for defining the study population at a site, and ways to develop distributions of individual risk for members of the study population. This paper focuses on the changes in the human exposure scenarios of the update method and presents an overview of the development of an exposure scenario.