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 dledbetter@genetics.emory.edu

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

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.

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,

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

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,72:918-930 Lese Martin et al. (2002) J Med Genet 39(10):734-740

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

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

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

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…

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

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

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

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

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

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

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

22q11 duplication 01.1727

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)

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.

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

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

Chromosome 13

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

Chromosome 10p Chromosome 10p

Chromosome 10q Chromosome 10q

Results # Cases Chromosome # Result 1 10 p-;q+ 2 13 or 21 q- 6 16 p+;q-

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

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

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.

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: 1 clone ~ 470 kb Average coverage in q11-q13: 1 clone ~150 kb

aCGH-15 Pilot Study Class II deletion Class I deletion

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

As normal as normal can be?

~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. - 465 kb median - 222 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.

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.