Jason Smith

Rare deletions at 16p13.11 predispose to a diverse spectrum of sporadic epilepsy syndromes.

Deletions at 16p13.11 are associated with schizophrenia, mental retardation, and most recently idiopathic generalized epilepsy. To evaluate the role of 16p13.11 deletions, as well as other structural variation, in epilepsy disorders, we used genome-wide screens to identify copy number variation in 3812 patients with a diverse spectrum of epilepsy syndromes and in 1299 neurologically-normal controls. Large deletions (> 100 kb) at 16p13.11 were observed in 23 patients, whereas no control had a deletion greater than 16 kb. Patients, even those with identically sized 16p13.11 deletions, presented with highly variable epilepsy phenotypes. For a subset of patients with a 16p13.11 deletion, we show a consistent reduction of expression for included genes, suggesting that haploinsufficiency might contribute to pathogenicity. We also investigated another possible mechanism of pathogenicity by using hybridization-based capture and next-generation sequencing of the homologous chromosome for ten 16p13.11-deletion patients to look for unmasked recessive mutations. Follow-up genotyping of suggestive polymorphisms failed to identify any convincing recessive-acting mutations in the homologous interval corresponding to the deletion. The observation that two of the 16p13.11 deletions were larger than 2 Mb in size led us to screen for other large deletions. We found 12 additional genomic regions harboring deletions > 2 Mb in epilepsy patients, and none in controls. Additional evaluation is needed to characterize the role of these exceedingly large, non-locus-specific deletions in epilepsy. Collectively, these data implicate 16p13.11 and possibly other large deletions as risk factors for a wide range of epilepsy disorders, and they appear to point toward haploinsufficiency as a contributor to the pathogenicity of deletions.

Nucleolar Proteins Suppress Caenorhabditis elegans Innate Immunity by Inhibiting p53/CEP-1

The tumor suppressor p53 has been implicated in multiple functions that play key roles in health and disease, including ribosome biogenesis, control of aging, and cell cycle regulation. A genetic screen for negative regulators of innate immunity in Caenorhabditis elegans led to the identification of a mutation in NOL-6, a nucleolar RNA-associated protein (NRAP), which is involved in ribosome biogenesis and conserved across eukaryotic organisms. Mutation or silencing of NOL-6 and other nucleolar proteins results in an enhanced resistance to bacterial infections. A full-genome microarray analysis on animals with altered immune function due to mutation in nol-6 shows increased transcriptional levels of genes regulated by a p53 homologue, CEP-1. Further studies indicate that the activation of innate immunity by inhibition of nucleolar proteins requires p53/CEP-1 and its transcriptional target SYM-1. Since nucleoli and p53/CEP-1 are conserved, our results reveal an ancient immune mechanism by which the nucleolus may regulate immune responses against bacterial pathogens.

Haptotactic Gradients for Directed Cell Migration: Stimulation and Inhibition Using Soluble Factors

A recently developed technique for the measurement of cell migration on surface bound gradients was used to assay the behavior of microvascular endothelial cells on a range of fibronectin gradient slopes in the presence of soluble promoters and inhibitors of chemotaxis. Directional microvascular endothelial cell migration was shown to increase with increasing gradient slope with no significant change in cellular persistence time or random cell speed. Uniformly distributed soluble chemotactic factor in the hMEC growth media enhanced directional migration. The addition of migration-inhibiting LY294002 eliminated the directional component of cell migration at a 5 microM dosing. These experiments broaden the understanding of the directional nature of cell motion and present a reliable system for the quantitative study of cell migration in complex conditions in vitro.

The optimization of quill-pin printed protein and DNA microarrays

Microarray density has been optimized as a function of substrate wettability and composition of the printing buffer. Features were printed across contact angle gradients to determine the effect of surface wettability on feature spreading. Feature size increased by nearly 50% and feature geometry transitioned from square to round as the array progressed from hydrophobic to hydrophilic for a wide range of pin sizes. The viscosity of water-glycerol and water-sucrose buffers were varied from 1-160 cP resulting in increases of up to 160% in printed feature size. Viscosity values were chosen to represent the potential working range for protein and DNA solutions. The amount of observed spread was determined to be independent of the size of the printing pin. Spreading diagrams that predict the spread in feature size beyond the size of the printing pin as a function of the water contact angle of the substrate and the viscosity of the printing buffer have been generated. Solutions containing proteins and DNA of various sizes have also been characterized, printed and demonstrated to fall within the trends observed in the buffer. Finally, initial work has been conducted using a planar waveguide system to study the kinetics of microarray performance.