International Endogene Study finds strong evidence of susceptibility loci for endometriosis at two genomic regions

1
BLOOD URINE COLLECTION
Set of blood collection tubes
1 x 4.5 ml. Citrate vacutainer Citrated plasma
2 x 9 ml. EDTA vacutainer DNA isolation and
EDTA-plasma
1 x 2 ml. EDTA vacutainer Measurement of HbA1c,
hemoglobin and hematocrit
2 x 9 ml. Heparin vacutainer RNA isolation
Urine collection set 2 x vacutainers, 1 x
adaptor Collection of urine
1 x 9 ml. Heparin vacutainer Isolation of
Leucocytes and heparin plasma
2
BLOOD URINE COLLECTION
1 x 4.5 ml. Citrate vacutainer
1 x 2 ml. EDTA vacutainer
2 x 9 ml. EDTA vacutainer
2 x 9 ml. Heparin vacutainer
1 x 9 ml. Heparin vacutainer
2 x 10 ml. Urine
Store in melting ice during transport
Store in melting ice during transport
Tube A Freeze within 1 hr. Store in dry-ice
during transport Tube B Add challenger within
1 hr. Store at 37C during transport
Store at room temperature during transport
3
EDTA DNA PLASMA
BLOOD HANDLING
2 x 9 ml. EDTA vacutainer

Store in melting ice during transport
Within 6 hr.
Centrifuge 20 min. 2000 G and 4C

Vacutainer with red cells, buffy- coat and rest
plasma
Collect plasma in plastic tube
Vortex shortly
lt1 hr RT
Store at -20 C
Division of plasma
18 Subsamples of 500 µl
lt1 hr RT
Snap-freezen
Store at lt-30 C
Methanol / Dry-ice
4
Heparine RNA (rest challenge)
BLOOD HANDLING
2 x 9 ml. Heparine vacutainer
TUBE A
TUBE B
lt1 hr RT
lt1 hr RT
Add challenger
3 subsamples of 3 ml
Store at 37C during transport
Snap-freezen
After 6 hr. (exactly)
Methanol / Dry-ice
3 subsamples of 3 ml
Store in dry-ice during transport
Snap-freezen
Methanol / Dry-ice
After arrival at TNO Store atlt-60 C
Store atlt-60 C
5
Year 2004 to Nov only
6
Array Based Genotyping Costs must reduce further
7
AGRF Affymetrix Chip Genotyping Concordance and
fail rates

• Concordance comparison of samples with SNP fail
  rate of lt 3
• Samples tested were an MZ twin pair, plus
  parents (S3 and S4).
• Genomic, Buccal and MDA Genomic replicates were
  tested for each individual.
• Sample MZ 2 Buccal was tested twice.
• Fail - no call for one or both samples

8
Association Analysis

• Sharing between unrelated individuals
• Disease alleles originate in common ancestor
• High resolution
• Recombination since common ancestor
• Large number of independent tests
• Powerful if assumptions are met
• Same disease haplotype shared by many patients
• Sensitive to population structure

9
Single Nucleotide Polymorphisms (SNP)
GGCTTCAGAATGGCCGGCTTCAAAATGGCC

• Single base changes
• Human SNPs 9,856,125
• - Validated SNPs 4,540,241
• Frequency 1 every 400 bp
• Can cause functional changes

10
QIMRs Sequenom MassARRAY Installation (CCRC-E
floor)
11
Multiplex Analysis
12
SNP Genotyping

• Minimum Finished Genotypes (gt99)
• Quality of DNA
• Measure concentrations
• Dispense in large volumes
• Quality of Assays
• Even peak heights
• Test for Hardy-Weinberg equilibrium
• Analysis of SNP data is particularly sensitive to
  assay problems
• Genotype failures are not random
• Heterozygous individuals fail most often
• all SNP typing platforms
• Error frequency of 0.11
• 3268 DNA samples typed twice
• 159 pairs of MZ twins - No discordant genotypes

13
Twelve-Plex Genotyping
14
Whole Genome Association

• Use DNA pooling to greatly reduce amount of
  genotyping
• Possible now but reduced power
• Use haplotypes to reduce number of SNPs that have
  to be genotyped
• Haplotype blocks HapMap project
• Massive parallel genotyping
• Costs must reduce further