Download Ledbetter Presentation 8/15/05

Genomic microarrays: Applications
to gene discovery and molecular
karyotype
August, 2005
David H. Ledbetter, Ph.D.
Department of Human Genetics
Emory University
[email protected]
Human Telomere Structure
centromere
(T2AG3)n
3 -20 kb
Unique DNA
Subtelomeric Region
100 - 300 kb
Martin and Ledbetter
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
Molecular Ruler
1 Mb contig
1 clone/500 kb to 5 Mb
(T2AG3)n
Subtelomeric Region
Telomere probe
Unique DNA
17p: Genotype/Phenotype
Normal
Miller-Dieker
ABR
1 Mb
tel – 68F18
CRK I&II
14-3-3
1.5 Mb
250 kb
RPA1
2.0 Mb
1029F21
HIC1
2.5 Mb
500 kb
LIS1
2.8 Mb
ILS
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
 Need
intensive
for a whole genome approach to
submicroscopic deletions/duplications
which is amenable to automation
Array CGH
Patient DNA
Genomic
Clones
Gain
Loss
Resolution
= clone size
~ 100 kb
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
1Mb
Array Examples
Y
X
Patient DNA
(male)
Control DNA
(female)
Gain = green
Loss = red
Normal = gray
X
X
X
Y
X
Normal
• Each clone is spotted multiple times
for reproducibility
• Clones from the same chromosomal
region are placed apart from each
other
Array Examples
Y
X
Patient DNA
(male)
Control DNA
(female)
X
X
X
Y
X
Normal
Gain = green
Loss = red
Trisomy 21
Ratio Display: female, 16p+; 17p-
X
Y
MR - Blinded Study Results
FISH
#
Detected
by array
1p deletion
1
yes
6q deletion
1
yes
9q deletion
1
yes
16p duplication
3
yes (3)
17p deletion
2
yes (2)
22q deletion
5
yes (5)
Derivative chrom.
4
yes (4)
ALL 17 IMBALANCES WERE
IDENTIFIED USING CGH-ARRAYS
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
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
4 Mb
22q tel deletion
4 Mb
1p tel deletion
6.5 Mb
16p tel duplication
10 Mb
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
Subtelomeric repeats
Unique DNA
(TTAGGG) n
Chromosome 13
Phenotype – Chromosome 10 Marker
 Routine
prenatal for AMA
 At
1 year of age patient exhibited slightly
delayed expressive language skills
 At
 At
19 months, oral motor dyspraxia noted
2 years expressive language delays
resolving
Chromosome 10p
Chromosome 10p
Chromosome 10q
Chromosome 10q
Results
# Cases Chromosome
#
1
10
Result
p-;q+
2
13 or 21
q-
6
14 or 22
q-
1
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
12 mm
2,500 clones; 1.4 Mb
» UCSF, British Columbia Genome
Center,…
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
Homebrew clones
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

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?
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)
76
255
Avg. - 465 kb
median - 222 kb
150 kb – 2 Mb
11
12.4
# of Loci
Length
Avg. diff. b/t
any 2 indiv.
*ROMA = Representational Oligonucleotide Microarray Analysis)
• Only 11 loci in common (within 1 Mb)
• In both studies, half were observed in >1 indiv.
Acknowledgements
Emory University
Christa Lese Martin,
Ph.D.
Vysis/Abbott:
Kim Wilber
Walter King
Teresa Ruffalo
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.