1. Department of Human Genetics
Genomic microarrays: Applications to gene discovery and molecular karyotype
August, 2005
David H. Ledbetter, Ph.D.
Department of Human Genetics
Emory University

2. Human Telomere Structure
centromere
(T2AG3)n
3 -20 kb
Unique DNA kb
Subtelomeric Region
Martin and Ledbetter 2

3. Telomere Abnl. Frequency in MR
Biesecker, Am. J. Med. Genet. 107: , 2002
Reviewed 14 studies, 1,718 patients with MR
6% overall abnormal results (range 2-29%)
50% of abnormalities inherited from balanced translocation parent
Conclusion: G-banding alone is insufficient to identify clinically significant segmental aneusomy. Additional molecular cytogenetic technologies are needed.

4. Subtelomere Analysis on 12,000 Cases
Britt Ravnan, James Tepperberg, Christa Martin
Genzyme, LabCorp and U. Chicago
>12,000 cases examined
3.2 % abnormals identified
0.4% polymorphisms, benign variants
2.8% clinically significant
In collaboration with Britt Ravnan at Genzyme Genetics and Jim Tepperberg at LabCorp,

5. Molecular Ruler 1 Mb contig 1 clone/500 kb to 5 Mb (T2AG3)n
This schematic illustrates a molecular ruler for one telomere region that incorporates multiple clones that span the most distal 5 Mb region.
For the most distal one megabase, it is useful to have a contig of overlapping clones so that smaller rearrangements can be characterized.
Then, for the remaining 4 Mb, one clone per every 500 kb is used.
(T2AG3)n
Subtelomeric Region
Telomere probe
Unique DNA 2

6. 17p: Genotype/Phenotype
Normal
Miller-Dieker
ILS
tel – 68F18
ABR
1029F21
CRK I&II
14-3-3
RPA1
LIS1
HIC1
2.8 Mb
2.5 Mb
2.0 Mb
1.5 Mb
500 kb
250 kb
1 Mb
13 genes can be deleted without phenotype
17p
Cardoso et al. (2003) Am J Hum Genet,72:
Lese Martin et al. (2002) J Med Genet 39(10):

7. Limitations of Telomere FISH
Does not assay whole genome
Labor intensive
Need for a whole genome approach to submicroscopic deletions/duplications which is amenable to automation

8. Array CGH Patient DNA Gain Resolution = clone size ~ 100 kb Loss
Genomic
Clones
Gain
Resolution
= clone size
~ 100 kb
Loss
Control DNA
Pinkel et al., Nat Genet (1998), 20(2):207-11

9. CGH array format GenoSensor™ Array 300 (N = 287)
Telomere array (N = 165)
Each telomere
Five X and two Y chromosome clones
“Molecular ruler” on 1p, 16p, 17p, and 22q.
TEL
CEN
5.0 Mb
1.5 Mb
2.0 Mb
2.5 Mb
1Mb

10. Array Examples Patient DNA (male) Control DNA (female) Gain = green
Loss = red
Normal = gray
Normal
Each clone is spotted multiple times for reproducibility
Clones from the same chromosomal region are placed apart from each other
MALE POC
THIS NEXT SLIDE SHOWS ALL OF OUR RESULTS…

11. Array Examples Patient DNA (male) Control DNA (female) Gain = green
Loss = red X X Y X
Normal
MALE POC
THIS NEXT SLIDE SHOWS ALL OF OUR RESULTS…
Trisomy 21

12. Ratio Display: female, 16p+; 17p-X Y

13. MR - Blinded Study Results# 1 3 2 5 4
Detected by array
yes
yes (3)
yes (2)
yes (5)
yes (4)
FISH
1p deletion
6q deletion
9q deletion
16p duplication
17p deletion
22q deletion
Derivative chrom.
ALL 17 IMBALANCES WERE
IDENTIFIED USING CGH-ARRAYS

14. Case 1: 8p deletion Phenotype: MR, hypopigmentation 8p tel
Ratio = 0.51
8p tel
Ratio = 0.52
Phenotype: MR, hypopigmentation

15. Case 2: 1ptel deletion with size of 4 Mb
Multiple 1ptel clones from telomere to 4 Mb
Phenotype: MR, obesity

16. Telomere Molecular Rulers
aCGH results
Size estimation
22q tel deletion
130 kb
17p tel deletion
1.9 Mb
16p tel duplication, 17p deletion
3.5 Mb for 16p, ~2.7 Mb for 17p
1p tel deletion
4 Mb
16p tel duplication
6.5 Mb
10 Mb

17. Patient DNA (male), Control DNA (female)
22q11 duplication
22q
Ratio: 1.4
V300 ARRAY
Patient DNA (male), Control DNA (female)
Gain = green, Loss = red

18. 22q11 duplication

19. Microduplications Identified by Array
4qtel duplication:
MR, seizures, cerebellar atrophy
Phenotypically normal mother has same duplication.
10qtel duplication:
Microcephaly, spasticity
Phenotypically normal father has same duplication.
10qtel duplication (2 clones):
Microcephaly, autism (parents pending)
22q11 duplication:
bilateral colobomas, DD, seizures, left ptosis (Cat Eye)

20. MR Blinded Study Conclusions
Demonstrates the sensitivity and accuracy of CGH-arrays since we detected 100% of all imbalances (n=17) identified by FISH;
Identified 4 small duplications not detectable by metaphase FISH, at least one clinically significant.
Potential for a more sensitive and cost-effective test for telomere and genome-wide screening since the assay is automatable.

21. Pericentromeric “Rulers”
Development of Centromere Molecular Rulers for identification and “calibration” of supernumerary marker chromosomes
Most proximal unique genomic clone to each pericentromeric “junkyard”
1 Mb contig plus 1 clone every 500 kb to 5 Mb away from pericentromeric region
Validated as unique FISH signal, map position and order

22. Pericentromeric regionα -
satellite (
TTAGGG) n
Pericentromeric region
Unique DNA
Subtelomeric repeats

24. Chromosome 13

25. Phenotype – Chromosome 10 Marker
Routine prenatal for AMA
At 1 year of age patient exhibited slightly delayed expressive language skills
At 19 months, oral motor dyspraxia noted
At 2 years expressive language delays resolving

26. Chromosome 10p
Chromosome 10p

27. Chromosome 10q
Chromosome 10q

28. Results # Cases Chromosome # Result 1 10 p-;q+ 2 13 or 21 q- 616
p+;q-

29. Array Formats LOW RESOLUTION
Targets only clinically relevant loci and clones are not spaced evenly across genome
Commercial
Vysis/Abbott, Spectral Genomics,…
Home Brew
Baylor, Signature Genomics,…
400 clones

30. Array Formats HIGH RESOLUTION Range from 1-3 Mb spacing
to complete genome tiling path
consisting of >32,000 clones!
Commercial
Spectral Genomics,…
Home Brew
UCSF, British Columbia Genome Center,…
12 mm
2,500 clones; 1.4 Mb
Array image from UCSF website

31. Array Formats – High Resolution
 coverage
32,855 BACs
~79 kb resolution
Developed by:
BC Cancer Agency
Genome Sciences
Center
Krzywinski et al., Nucleic Acids Res (2004) 32(12):

32. aCGH-15 Pilot Study- Clone Selection
Tiling Path Clones
Chose the two re-array plates that had the highest number of clones in the 15q11-15q13 region. (Plates 2B1 and 3A1)
3A1 was a partially filled plate of 43 clones
135 tiling path clones total
Homebrew clones
36 clones at significant loci on chr. 15
Breakpoints
genes
segmental duplications
7 Sex Chrm. Clones
Total clones: 178
Average coverage on chr15: clone ~ 470 kb
Average coverage in q11-q13: 1 clone ~150 kb

33. aCGH-15 Pilot Study
Class II deletion
Class I deletion

34. aCGH-15 Pilot Study aCGH-15 Patient 1 Pervasive Developmental Delay
Phenotype suggestive of PW

35. As normal as normal can be?

36. ~150 kb every 1 Mb (50 kb detection size)
Sebat et al.
Iaftrate et al.
Technique
ROMA* - 20 indiv.
Array CGH - 55 indiv.
Resolution
~1 probe / 35 kb
(105 kb detection size)
~150 kb every 1 Mb (50 kb detection size)
# of Loci 76
255
Length
Avg kb
median kb
150 kb – 2 Mb
Avg. diff. b/t any 2 indiv. 11
12.4
*ROMA = Representational Oligonucleotide Microarray Analysis)
Only 11 loci in common (within 1 Mb)
In both studies, half were observed in >1 indiv.

37. Acknowledgements Vysis/Abbott: Emory University Kim Wilber
Walter King
Teresa Ruffalo
Emory University
Christa Lese Martin, Ph.D.
Andrew Wong, Ph.D.
Lorraine May, M.S.
David Johnson, B.S.
Devan Pressley, B.S.
Courtney Works, B.S.
Grant Support:
March of Dimes
NIH
Vysis/Abbott, Inc.