Genomic microarrays: Applications to gene discovery and molecular karyotype

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

2. (T2AG3)n 3 -20 kb Subtelomeric Region 100 - 300 kb Human Telomere Structure centromere Unique DNA Martin and Ledbetter

3. Telomere Abnl. Frequency in MR Biesecker, Am. J. Med. Genet. 107:263-266, 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

5. 1 Mb contig (T2AG3)n Subtelomeric Region 1 clone/500 kb to 5 Mb Telomere probe Unique DNA Molecular Ruler

6. Normal Miller-Dieker ILS 17p: Genotype/Phenotype 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 1 Mb 500 kb 250 kb 17p Cardoso et al. (2003) Am J Hum Genet,72:918-930 Lese Martin et al. (2002) J Med Genet 39(10):734-740

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 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 2.5 Mb 2.0 Mb 1.5 Mb 1Mb

10. Y X X X X Y X 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

11. Array Examples Y X X Patient DNA (male) Control DNA (female) Gain = green Loss = red X X Y X Normal Trisomy 21

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

13. MR - Blinded Study Results Detected by array yes yes yes yes (3) yes (2) yes (5) yes (4) # 1 1 1 3 2 5 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 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

17. 22q11 duplication 22q Ratio: 1.4 V300 ARRAY Patient DNA (male), Control DNA (female) Gain = green, Loss = red 01.1727

18. 22q11 duplication 01.1727

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 region Pericentromeric region Subtelomeric repeats Subtelomeric repeats Unique DNA Unique DNA ( ( TTAGGG) TTAGGG) α α - - satellite satellite n

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

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):3651-60.

32. 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 aCGH-15 Pilot Study- Clone Selection 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: 1 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. *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: 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.