LELAND STANFORD JUNIOR UNIVERSITY, THE
The Gastroparesis Clinical Research Consortium (GpCRC), is sponsored by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) with the goal of providing an infrastructure and database that will faciliate clinical, pathophysiological epidemiological, and therapeutic research in gastroparesis. Under its auspices, we are conducting a study entitled the "Pathological Basis of Gastroparesis", which aims to understand the morphological and molecular changes that are associated with this syndrome. Our ongoing study has provided valuable insight into the nature of some of these changes and has also suggested the need for additional techniques to build on these results. This supplemental proposal requests funds to accelerate the tempo of research we originally proposed, by facilitating the following experiments: (1) To examine the nature of the inflammation in the muscle layers of the stomach. Cytokine and chemokine analysis will be performed using a Luminex bead array system on tissue and serum samples. (2) To identify potential signatures of infection in the stomach of patients with gastroparesis.We will take advantage of the recently developed GreeneChip technology that uses a panmicrobial oligonucleotide microarray containing sequences for every known vertebrate viruses, bacteria, fungi, and parasites. For the analysis, 2X2X2 mm piece of fresh frozen biopsies will be collected and total RNA extracted from the same patient pool as above. (3) To identify gene expression patterns in gastroparetic samples. Genome-wide expression analysis will be performed using Agilent human whole genome microarrays containing 44,000 oligonucleotide probes, The genetic signature will be analyzed further to identify candidate molecules involved in the pathogenesis of gastroparesis as well as tested on new sample sets once the initial pilot study is completed. (4) To investigate genomic changes in patients with gastroparesis.We will use array-based comparative genomic hybridization (array CGH) to perform high-throughput, high-resolution, genome-wide screening of DNA copy number changes at both the chromosomal and subchromosomallevel on stored genomic material (DNA) on these patients, obtained from the NIH gastroparesis repository. We will also analyze DNA sequences to look for polymorphisms of genes such as a2A (adrenergic receptor), NET (norepinephrine transporter), SERT (serotonin transporter, SCL6A4), G-protein beta-3 receptor (Gn~3), nNOS, VIP and H01 genes or their promoters and others.