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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. (T 2 AG 3 ) n. 3 -20 kb. Subtelomeric Region. 100 - 300 kb. Human Telomere Structure.

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genomic microarrays applications to gene discovery and molecular karyotype
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

(T2AG3)n

3 -20 kb

Subtelomeric Region

100 - 300 kb

Human Telomere Structure

centromere

Unique DNA

Martin and Ledbetter

telomere abnl frequency in mr
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
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

molecular ruler

1 Mb contig

(T2AG3)n

Subtelomeric Region

1 clone/500 kb to 5 Mb

Telomere probe

Unique DNA

Molecular Ruler
slide6

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

limitations of telomere fish
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
slide8

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

cgh array format
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

array examples

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
array examples1
Array Examples

Y

X

X

Patient DNA (male)

Control DNA (female)

Gain = green

Loss = red

X

X

Y

X

Normal

Trisomy 21

mr blinded study results
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

case 1 8p deletion
Case 1: 8p deletion

8p tel

Ratio = 0.51

8p tel

Ratio = 0.52

Phenotype: MR, hypopigmentation

case 2 1ptel deletion with size of 4 mb
Case 2: 1ptel deletion with size of 4 Mb

Multiple 1ptel clones from telomere to 4 Mb

Phenotype: MR, obesity

slide17

22q11 duplication

22q

Ratio: 1.4

V300 ARRAY

Patient DNA (male), Control DNA (female)

Gain = green, Loss = red

01.1727

microduplications identified by array
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
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
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
slide22

Pericentromeric region

Pericentromeric region

Subtelomeric repeats

Subtelomeric repeats

Unique DNA

Unique DNA

(

(

TTAGGG)

TTAGGG)

α

α

-

-

satellite

satellite

n

phenotype chromosome 10 marker
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 10p

chromosome 10q
Chromosome 10q

Chromosome 10q

array formats
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 formats1
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
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.

slide32
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

slide33

aCGH-15 Pilot Study

Class II deletion

Class I deletion

slide34

aCGH-15 Pilot Study

aCGH-15

Patient 1

Pervasive Developmental Delay

Phenotype suggestive of PW

slide36

*ROMA = Representational Oligonucleotide Microarray Analysis)

  • Only 11 loci in common (within 1 Mb)
  • In both studies, half were observed in >1 indiv.
slide37

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.