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 thetreatment of neurological diseases.

• 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

ECT ProductsECT for Parkinson’s Disease

• Michael J. Fox foundation granted US $3 million to support a clinical “proof-of-concept” (May 2004)

• Identification of novel genes by use of bioinformatics

• Neublastin (GDNF family – potent neuroprotective effects)

Factor Products

• Scanning the human genome or assembled protein sets for different features of interest

A case study

• Search for Parkinson related gene(s)

• Affymetrix GeneChip experiments

• Fetal brain tissue

Parkinson DiseaseDegenerative central nervous system (CNS) disorder

Parkinson DiseaseLoss 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 dopamineproducing cells replace the dead ones.

Dopamin producing brain cells from aborted foetuses have beenoperated into the brain of parkinson patients and ín some casescured the disease. Brain tissue from approx 6 foetuses were needed.

Major ethical problems !

Search for a protein drug is the only valid option

Parkinson DiseaseDopamine 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 tissueMidbrain mesencephalon

Vm Dm+ Dopamine producingcells

- Dopamine producingcells

• Aborted feotus brain tissue – Karolinska hospital

• Feotus of age 6-10 weeks, 2 cases

Midbrain mesencephalon

Vm Dm

+ Dopamineproducingcells

- Dopamine producingcells

RNA purification + amplification

Affymetrix genechip analysis

Isolate the two samples (Vm/Dm)

Dopamine producing cells at the interface ?

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. Theprobes are normally not a part of a protein sequence.

Affymetrix probe

Blast

IPI protein sequence

Blast inferred

Unigene sequence (cDNA)5’ 3’

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”

BLATThe 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

BLATBlast Like Alignment Tool

• BLAT (2002)

• 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 Browserhttp://genome.ucsc.edu//

BLAT genome Browser

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

BLAT genome Browserhttp://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 BrowserZoom in ’base’

BLAT genome Browser”Browser”

TremblSwissProt 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