Gene Discovery & Genome Browsing Background – myself NsGene – DTU satellite Parkinson Disease (Affymetrix GeneChip) Analysis of fetal brain tissue Genome browsing Introduction to the UCSC BLAT browser

Background Thomas Nordahl Petersen Chemist, Ph.D protein Crystallography, University of Copenhagen Computational Scientist, SBI-AT Prediction of protein structure, secondary structure, fold recognition, homology modeling Bioinformatics - Gene discovery, NsGene Devolop novel cell and gene based products for the treatment of neurological diseases.

ECT Products ECT for Parkinson’s Disease Growth of cells in a capsule matrix The therapeutic protein be released directly in the relevant brain area Safe delivery across the blood-brain-barrier Michael J. Fox foundation granted US $3 million to support a clinical “proof-of-concept” (May 2004)

Factor Products Identification of novel genes by use of bioinformatics Neublastin (GDNF family – potent neuroprotective effects) Scanning the human genome or assembled protein sets for different features of interest

A case study Affymetrix GeneChip experiments Fetal brain tissue Search for Parkinson related gene(s) Affymetrix GeneChip experiments Fetal brain tissue

Parkinson Disease Degenerative central nervous system (CNS) disorder

Parkinson Disease Loss of dopamine producing brain cells

Parkinson’s Disease Dopamine from Substantia nigra activates neurons in Striatum/Basal ganglia Important for initiation of movement

Cure for Parkinson’s Disease ? Parkinson disease may be cured provided that new dopamine producing cells replace the dead ones. Dopamin producing brain cells from aborted foetuses have been operated into the brain of parkinson patients and ín some cases cured the disease. Brain tissue from approx 6 foetuses were needed. Major ethical problems ! Search for a protein drug is the only valid option

Parkinson Disease Dopamine producing cells Dopaminergic neurons can be found in the ventral part of the mesencephalon (VM) from approximately 6 weeks No dopaminergic neurons can be found in the neighbouring dorsal part (DM). Dopaminergic differentiation by use of GeneChips to compare the expression profiles of VM and DM

Fetal brain tissue Midbrain mesencephalon - Dopamine producing cells + Dopamine producing cells Vm Dm Aborted feotus brain tissue – Karolinska hospital Feotus of age 6-10 weeks, 2 cases

Midbrain mesencephalon Dm Vm - Dopamine producing cells + Dopamine producing cells Dopamine producing cells at the interface ? Isolate the two samples (Vm/Dm) RNA purification + amplification Affymetrix genechip analysis

GenePublisher (program by Steen Knudsen) Scale, normalize the Affymetrix GeneChip experiments A1 A2 A2 B1 B2 B2 P-value 319 315 314 44 48 38 1.26e-07 314 334 327 443 434 444 6.55e-05 1980 1974 1973 1801 1785 1763 6.77e-05 123 123 126 87 88 93 8.01e-05 103 101 104 77 78 73 0.000112 107 107 111 79 77 82 0.000124 128 123 117 189 184 196 0.000142 179 179 186 145 147 149 0.000191 78 77 79 86 87 87 0.000202 96 90 93 136 129 138 0.000215

Vulcano plot P-value Log2 Fold change

Assigning Affymetrix GeneChip probes to a protein sequence ~20.000 probes on each of the A/B Affymetrix chips. The probes are normally not a part of a protein sequence. Unigene sequence (cDNA) 5’ 3’ IPI protein sequence Blast Affymetrix probe Blast inferred

Internal database

Signal Peptide prediction

Conclusion – so far The most up-regulated genes include several ‘known’ genes like dopamine transporter (good positive control) The most interesting genes are the ‘unknowns’ that were up-regulated in Vm. Futher analysis is ongoing. Roland JR et al., Exp Neur (2006) Vol 198,2,427-437 “Identification of novel genes regulated in the developing human ventral mesencephalon”

BLAT The Blast Like Alignment Tool Smith-Waterman (1980) Local alignment (best alignment) FASTA (1988) Fast alignment BLAST-programs (1990,1997) Query seq versus an indexed database MegaBLAST (2000) Fast alignment of DNA sequences SSAHA (2001) Maps sequence reads to genome

BLAT Blast Like Alignment Tool Very fast searches (MySQL database) Handle introns in RNA/DNA alignments Data for more that 30 genomes (human, mouse, rat…) Exon Intron Exon Splice sites

BLAT genome Browser http://genome.ucsc.edu//

BLAT genome Browser Using a search term or position eg Chr1:10,234-11,567

BLAT genome Browser http://genome.ucsc.edu/

BLAT genome Browser Using a protein or DNA sequence

Blat genome Browser

BLAT genome Browser ”Details”

Blat genome Browser

BLAT genome Browser ”Browser”

BLAT genome Browser Zoom in ’base’

BLAT genome Browser ”Browser” Trembl SwissProt Hypothetical

BLAT genome Browser ”Description & Page Index”

BLAT genome Browser ”Description & Page Index”

Blat Genome Browser ”tracks”

BLAT genome Browser ”SNPs”

BLAT genome Browser ”SNPs”

Gene Sorter

Gene sorter