Title: Probing the Genome using scFISH Single Copy Fluorescence In Situ Hybridization
1 Sequence-based In Situ Detection of Chromosomal
Abnormalities at High Resolution -
Probing the Genome with scFISH
Joan HM Knoll, PhD, FACMG, FCCMG University of
Missouri-Kansas City School of Medicine
2The Paradigm
- Prenatal, postnatal and neoplastic chromosomal
abnormalities - are increasingly being identified or confirmed by
molecular - cytogenetics (ie. F.I.S.H. or fluorescence in
situ hybridization). - Nucleic acid probes are directed to
rearrangements or aneuploidies of specific genes
or chromosomal intervals that have been
implicated in the clinical defects. - Therapies in the future will be tied directly to
DNA diagnostic - technologies that stratify patients into risk
categories defined - by chromosomal abnormalities.
3Molecular Cytogenetic Test FISH
Complementary nucleic acid and chromosomal target
DNA bind noncovalently binding detected by
fluorescence.
4Applications of FISH
- Clinical detection of chromosomal gain, loss,
origin, cryptic translocations, microdeletions,
etc - constitutional - prenatal, pediatric, adult
- acquired - neoplasia
- Research gene mapping, chromatin structure and
organization, etc
5Availability of Locus Specific Commercial Probes
Inherited abnormalities Subtelomeric
regions Acquired abnormalities
6Commercial Probes Properties
- Selected for frequent abnormalities (limited in
number) - Recombinant clones - defined experimentally
(large and generally not sequenced) must be
obtained and propagated, delaying the analysis - Validated to rule out cross-hybridization to
other genomic targets - Yield large hybridization signals due to long
chromosomal target length - Large size precludes precise breakpoint
localization
7Conventional Fluorescent In Situ
Hybridization Procedure
Genomic probe
single stranded DNA
Single copy gene sequences
double stranded DNA
repetitive sequences
Excess of Denatured Competitor DNA
(Cot 1 DNA)
Labeled and denatured probe DNA
Preannealing
Hybridization (repetitive sequences are disabled)
Detection by fluorescence
Probe
Chromosome DNA on microscope slide
8 Nonspecific Hybridization without
Cot 1 DNA Blocking
9Conventional FISH Chromosome X Probes
Green DXZ1 Red KAL1 cosmid clones
10Sequence-based scFISH probes Properties
- Developed for both common and rare
abnormalities - Uses available human genome sequences (Public
Consortium Celera Genomics databases) - Produced without library construction,
screening, or propagation of recombinant DNA
clones - Shorter unique sequence probes
- do produce smaller hybridization signals,
- but enable precise breakpoint delineation
- generally do not cross hybridize to other
targets
US and International patents pending
11Step 1 Obtain sequence of interest
- Delineate chromosomal region containing gene(s)
associated with disorder, - Obtain mRNA sequence of gene(s),
- Compare with genomic sequences to obtain
corresponding complete gene and adjacent
sequences.
Example
DiGeorge, Shprintzen, Velocardiofacial Syndromes
Chromosome 22 genomic sequence
HIRA
OMIM No. 188400
ZNF74
Genes GenBank (mRNA) HIRA X7529 6 ZNF74
X71623
12Step 2 Deduce locations of single copy intervals
- Computer program compares genomic sequence (gt100
kb) with database of (440) repetitive sequence
families. - Determine the locations of repetitive genetic
elements in genomic sequence. - Align results with gene sequence.
cDNA
Genomic
Repetitive sequences
Single copyintervals
13Step 3 Amplify and purify single copy sequences
- Sort sequence intervals by decreasing lengths,
- Computer-aided selection of primers for PCR
amplification of longest intervals, - Long PCR of gt2 kb fragments, isolate DNA
amplification products.
Iterate to maximize product length, annealing
temperature, GC content based on composition
1 2 3 4 kb
14Sizes Locations of Single Copy Intervals in 3
Chromosomal Regions
22q11.2
15q11.2
1p36.3
15Genomic Interval Length Needed to Develop Probes
Determined from the locations of single copy
intervals on a random sample of chromosome 21
and 22 sequences. Sampling rate was 0.5. Rogan,
Cazcarro, Knoll, Genome Research 2001.
16Applications of scFISH Probes
- Detect common abnormalities
- Examine phenotype-genotype relationships
- Identify locations of chromosome translocation,
inversion and deletion breakpoints - Delineate paralogous sequence families and
exploit these sequences in detection of
rearrangements - Determine previously unknown repetitive sequences
- Define extent of cryptic rearrangements
characterize sequences involved in rare or
private chromosomal rearrangements - Explore chromosome structure
17(No Transcript)
18Phenotype-Genotype Relationships
Examples - Detection of small IC deletions in
Angelman and Prader-Willi syndromes - Detection
of atypical deletions in Smith-Magenis syndrome
19ANGELMAN and PRADER-WILLI SYNDROMES
- AS and PWS are clinically distinct syndromes
- localizes to chromosome 15q11.2q13
- maternal genetic information is absent in AS
- paternal information is absent in PWS
- frequency 1/20,000
AS
Etiology PWS AS Deletion 70 70
Uniparental disomy 25 5 Other
5 25
PWS
20PRADER-WILLI and ANGELMAN SYNDROMES
MAGEL2
Karyotype 46,XY,del(15)(q11.2q13).ish
del(15)(q11.2q13)(MAGEL2-)
21CHROMOSOME 15q11.2q13 AS/PWS REGION
PWS IC deletion (SRO)
Common deletion
Nicholls et al, 1989 Knoll et al, 1989 Gregory et
al, 1990 Saitoh et al, 1996
22Detection of the PWS Imprinting Center by scFISH
scFISH/FISH detection rate PWS 99 of
abnormalities AS 80 of abnormalities (not
UBE3A mutations)
includes replication timing FISH assay for UPD
(White et al. 1996).
scFISH IC probes potentially offer an alternative
to PCR-based DNA methylation analysis.
Probes PWS-SRO, MAGEL2
23Localization of scFISH probes on Ensembl
reference sequence
Complete probe listing with hyperlinks in Knoll
and Rogan, Amer J Med Genetics, in press.
24SMITH-MAGENIS SYNDROME
Clinical findings (common) Distinct facies
(brachycephaly,mid-face hypolasia, broad nasal
bridge), brachydactyly, short stature, hoarse
voice, MR, infantile hypotonia, eye problems,
pain insensitivity, sleep disturbances,
etc. Behavioral problems - Aggressive, excitable,
biting, skin picking, nail removal, etc. Other
less common features - Seizures, cardiac defects,
cleft/lip palate, scoliosis, etc. Etiology 95
have del(17)(p11.2)
25Chromosome 17p11.2 Smith-Magenis Region
Common interstitial deletion involving meiotic
mispairing of SMS REP paralogs Juyal et al,
1996 Potocki et al, 1998
26Atypical Deletion in Smith-Magenis Syndrome
17
Deletion FLI1 probe
Nondeletion ADORA2B probe
27Chromosome 17p11.2 Smith-Magenis Region
Our patient
Deleted
Intact
28Delineation of Translocation Breakage/Deletion
Intervals Chronic Myelogeneous Leukemia (CML)
- 1/100,000 people per year
- Most have t(922)
- Disrupts ABL1 oncogene on chromosome 9 and BCR
region on chromosome 22 - Occurs in all cell lineages
- Chronic, accelerated and blast phases
29Chronic Myelogenous Leukemia (CML)
9
22
Karyotype 46,XX,t(922)(q34q11)
30Sizes and Locations of Single Copy Intervals in
BCR and ABL1 Genes
Chromosome breakage region
31Chronic Myelogenous Leukemia and
t(922)(q34q11.2)
der(22)
9
ABL1, 5-probe cocktail Ex1b, IVS1b IVS3,
IVS4-6, IVS11
ABL1, 3-probe cocktail IVS3,
IVS4-6, IVS11
der 9 der 22 normal 9
normal 9
der 22
normal 9
32Single Copy Intervals (? 1500 bp) between the ASS
ABL1 Genes on Chromosome 9q34
bp
cen
tel
Patients with large deletions (ASS-ABL1) have
poor prognosis. What about smaller deletions?
scFISH permits detection of smaller deletions.
33Breakpoint Delineation Using scFISH Probe Clusters
One possible strategy.
Translocates to chromosome B
Chromosome A
1
2 3 4 5
6 7 8 9
Probe
tel
cen
Chromosome break
Probe clusters labeled in
Scale
First hybridization
10 kb
Second hybridization
Third hybridization . . .
Inferred breakpoint interval
34Breakpoint Delineation Using scFISH Probe
Clusters
1
2 3 4 5
6 7 8 9
Probe
cen
tel
Probes 1-9
Pattern
der(B)
der(A)
B
A
der(B)
der(A)
1-5
B
A
der(B)
der(A)
6-9
B
A
35Strategy for Detecting Chromosome 9q34 Deletions
by scFISH using Minimal of Hybridizations
1 to 5 hybridizations necessary to classify
molecular deletion subclass
Cen-ASS-FIB-FBP3-PRDM12-RRP4-ABL1-Tel
36Identification of Chromosome Rearrangements with
Paralogous Sequence Probes EXAMPLE Acute
Myelogenous Leukemia M4 with inv(16)(p13q22) WHY
study it? - presence confers a good
prognosis - often difficult to detect by
routine cytogenetics - confirm by FISH Paralog
member of gene family in same genome (gt95
homology)
37Acute Myelogenous Leukemia (AML M4)
Karyotype 46,XX,inv(16)(p13q22)
16
38Sizes and Locations of Single Copy Intervals in
Genes Detected in Inv(16)(p13q22) AML-Type M4
39scFISH with Paralogous Sequence Family from
chromosome 16p (PM5 Probe)
cell 2
cell 1
normal
inv(16)(p13q22)
Paralogous sequence probe splits signals in
inv(16). Multiple targets produce brighter
hybridizations.
40Delineation of Cryptic Rearrangements at
Chromosomal Ends
Why? Up to 10 of patients with idiopathic MR
have subtelomeric deletions using commercial
probes. Problem Commercial probes may not
detect hemizygosity adjacent to telomere due to
size and distance from telomere. Solution
Develop probes that are closer to chromosomal
ends.
41Locations of scFISH and Commercial Telomere
Probes
Prediction gt10 of IMR patients will have
terminal imbalances with scFISH probes.
42MONOSOMY CHROMOSOME 1P36 SYNDROME
CDC2L1
Karyotype 46,XY,del(1)(p36.1).ish
del(1)(p36.1)(CDC2L1-)
43Chromosome Structure/Organization
- Duplicons, paralogous sequences
- New repetitive sequences
- Chromosomal distribution of single copy intervals
- Different hybridization efficiency between
homologs (eg. Differential accessibility)
44Down Syndrome Critical Region Duplicon Probes
45New Repetitive Sequence Observed in DSCR4 Gene
(21q22.3)
DSCR4-1.9 kb
DSCR4
Low stringency wash 4 X SSC
High stringency wash 1 X SSC
Result Sequence is not related to rDNA, nor is
it from a sequence family adjacent to ribosomal
repeat (Gonzalez and Sylvester, 2000). Different
copy number/levels of conservation found on
acrocentric p arms and between individuals.
46Why does scFISH detect new repetitive sequences?
Genome sequence consists primarily of
euchromatic DNA centromeric, heterochromatic
and acrocentric short arm regions are often
difficult to assemble and propagate by
recombinant DNA techniques . . . . . .
resulting in some regions of the genome remaining
unsequenced. Thus, we anticipate that some
single copy probes containing undescribed
repeats may hybridize to unsequenced regions of
genome . . . . . . and these repeats may not
be represented in available human repetitive
family databases.
47Chromosome 22 Distribution and Sizes of Single
Copy Intervals
22.0
19.8
17.6
15.4
13.2
Length (Kbp)
11.0
8.8
6.6
4.4
2.2
0.0
0.0 3.4 6.8 10.2 13.6 17.0 20.4 23.8
27.2 30.6 34.0
Chromosomal coordinate (Mbp)
Centromere
Telomere
48Chromosome 22 Distances between Single Copy
Intervals (gt2.3 kb)
Q. Does the average distance between sc
intervals equal the expected value of 1 per 22
kb? A. No, observed is 1 per 10 kb, a finding
consistent with low density in heterochromatin.
Number of intervals
Max
Distance separating adjacent intervals
49Distribution of Distances Between Single Copy
Intervals (gt2.3 kb) Nonrandom at Extreme
Distances
untransformed
gt 2.3 kb sc intervals separated by by 50-1000
bp and by gt100kb more often than expected from a
random distribution.
Log10 Distance
50Future enhancements
- Automation of probe preparation
- Automation of metaphase scanning of scFISH probes
- Genome-wide single copy (sc) probe map and design
51Automated slide processing schema
Automated Fluorescence Microscope (CMH)
Daily
backup (CMH) UMKC-SICE
MU-Columbia (primary storage
(secondary storage) of XML)
Image analysis Image prioritization
microscope coordinates
Algorithm
and/or CMH Review by
parameter microscopist
refinement Selection of adequate images
Return image coordinates CMH Final
capture and optimization of individual images
Automated stage, camera,
filter wheel, Z-stack
52Summary
- scFISH rapidly generates probes from genomic
sequences (40 regions telomeres gt120 probes) - Allows faster characterization of chromosomal
abnormalities especially private rearrangements
both clinical and research utility - Permits chromosomal characterization at a much
greater resolution than previously possible - Provides new information about the genome new
repetitive sequences, chromosome structure
duplicons, accessibility - MAKES THE HUMAN GENOME SEQUENCE ACCESSIBLE AND
USEFUL TO THE CYTOGENETICIST!
53 Collaborations/Acknowledgements Computationa
l Molecular Biology, Automation Pete Rogan,
PhD, CMH Cytogenetics Specimens Janet Cowan,
PhD, NEMC Linda Cooley, MD, CMH Wendy
Fletjer, PhD, Esoterix, TN Val Lindgren, PhD,
UI Diane Persons, MD, KUMC Sharon Wenger, PhD,
WVU Daynna Wolff, PhD, MUSC Current Technical
Staff Patrick Angell, Angela Marion, Camille
Marsh, Patricia Walters Financial Support
National Cancer Institute - NIH Patton
Charitable Trust Foundation KB Richardson
Research Foundation Hall Foundation National
Science Foundation