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
The 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.
Molecular Cytogenetic Test: FISH
Complementary nucleic acid and chromosomal target DNA
bind noncovalently; binding detected by fluorescence.
Applications 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
Availability of Locus Specific Commercial Probes
Inherited abnormalities
Subtelomeric regions
Acquired abnormalities
Commercial 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
Conventional Fluorescent In Situ Hybridization:
Procedure
Genomic probe:
single stranded DNA
Single copy gene sequences
double stranded DNA
repetitive sequences
Labeled and denatured probe DNA:
+
Excess of Denatured Competitor DNA:
(Cot 1 DNA)
Preannealing
Hybridization
(repetitive sequences are disabled)
Detection by fluorescence
Probe
Chromosome DNA on microscope slide
Nonspecific Hybridization without
Cot 1 DNA Blocking
Conventional FISH: Chromosome X Probes
Green = DXZ1; Red = KAL1; cosmid clones
Sequence-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
OVERCOMES LIMITATIONS OF COMMERCIAL PROBES
*US and International patents pending
Step 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
Genes
HIRA
ZNF74
GenBank (mRNA)
X75296
X71623
ZNF74
Step 2: Deduce locations of single copy
intervals
•Computer program compares genomic sequence (>100 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
Step 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 >2 kb fragments, isolate DNA amplification
products.
1 2 3 4 kb
Iterate to maximize:
product length,
annealing temperature,
GC% content based on
composition
Sizes & Locations of Single Copy Intervals in 3 Chromosomal Regions
22q11.2
15q11.2
1p36.3
Genomic 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.
Applications 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
C atalogu e of S elected D isord ers D etected b y scF IS H
M o n o so m y 1 p 3 6
W o lf-H irsch o rn S x
C ri-d u -C h at S x
M yelo d ysp lastic S x
W illia m s S x
L an g er-G ied eo n S x
C M L (C h ro n ic M y elo g en o u s L eu k em ia)
A L L (A u cte L y m p h o cytic L eu k e m ia)
T riso m y 1 3 (Z IC 2 )
P rad er-W illi/A n g elm an S x
In v erted D u p licatio n 1 5 S x
A M L -M 4 (A cu te M yelo g en o u s leu k em ia -M 4 )
R u b en stein -T ayb i S x
C h arco t-M arie-T o o th D isease T yp e 1 A
S m ith - M ag en is S x
M iller-D iek er S x
T riso m y 1 8 (G A L R 1 )
A lag ille S x
D ow n Sx
D iG eo rg e/ V C F S S x
K allm an S x / T u rn er S x
C h ro m .
1
4
5
5
7
8
9 ;2 2
1 2 ;2 1
13
15
15
16
16
17
17
17
18
20
21
22
X /X p 2 1 .1
Phenotype-Genotype Relationships
Gain or loss of individual genes can be examined due to the
high-density and small size of scFISH probes.
Examples:
- Detection of small IC deletions in Angelman and
Prader-Willi syndromes
- Detection of atypical deletions in Smith-Magenis
syndrome
ANGELMAN and PRADER-WILLI SYNDROMES
• AS and PWS are clinically
distinct syndromes
•localizes to chromosome
15q11.2q13
•maternal genetic
information is absent in AS
AS
•paternal information is
absent in PWS
•frequency: ~1/20,000
PWS
Etiology:
PWS
AS
Deletion
Uniparental disomy
Other
~70%
~25%
~5%
~70%
~5%
~25%
PRADER-WILLI and ANGELMAN SYNDROMES
*
MAGEL2
Karyotype: 46,XY,del(15)(q11.2q13).ish del(15)(q11.2q13)(MAGEL2-)
CHROMOSOME 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
Detection 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 PCRbased DNA methylation
analysis.
Probes: PWS-SRO, MAGEL2
Localization of scFISH probes on Ensembl reference sequence
Chromosome
/Disorder
Gene
Interval
Cytogenetic nomenclature
15/PraderWilli,
Angelman
Sx
IC/SNRPN
IVS 5-Exon
u1B-IVS 3
ish del(15)(q11.2q11.2)(IC/
SNRPN-)
9/CML
ABL1
Exon 1b-IVS
1b
ish t(9;22)(q34;q11.2)(ABL
st)
16/AMLM4
PLA2G10
PKD
PM5
IVS 3
IVS 12-Exon
15
~100 kb
upstream
ish
inv(16)(p13q22)(PLA2G10
mv, PKD mv, PM5 sp)
Complete probe listing with hyperlinks: in Knoll and Rogan, Amer J Med Genetics, in press.
SMITH-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)
Chromosome 17p11.2: Smith-Magenis Region
Common interstitial deletion involving meiotic mispairing of SMS REP paralogs; Juyal et al, 1996;
Potocki et al, 1998
Atypical Deletion in Smith-Magenis Syndrome
17
Deletion* : FLI1 probe
Nondeletion: ADORA2B probe
Chromosome 17p11.2: Smith-Magenis Region
Our patient:
Deleted
Intact
Delineation of Translocation Breakage/Deletion
Intervals : Chronic Myelogeneous Leukemia (CML)
•1/100,000 people per year
• Most have t(9;22)
•Disrupts ABL1 oncogene on chromosome 9
and BCR region on chromosome 22
• Occurs in all cell lineages
•Chronic, accelerated and blast phases
Chronic Myelogenous Leukemia (CML)
9
22
Karyotype: 46,XX,t(9;22)(q34;q11)
*By conventional FISH, about 10% of patients also have a deletion on
chromosome 9 of sequences upstream of ABL1 (Berens et al, 2000;
Sinclair et al, 2000).
Sizes and Locations of Single Copy Intervals in BCR and ABL1 Genes
Chromosome breakage region:
Chronic Myelogenous Leukemia and t(9;22)(q34q11.2)
der(22)
9
ABL1, 3-probe cocktail:
IVS3, IVS4-6, IVS11
der 22
normal 9
ABL1, 5-probe cocktail:
Ex1b, IVS1b IVS3, IVS4-6, IVS11
der 9
der 22
normal 9
normal 9
Single Copy Intervals ( 1500 bp) between the ASS & ABL1
Genes on Chromosome 9q34
ASS
FBP3
PRDM12 RRPR4
ABL
bp
cen
tel
Patients with large deletions (ASS-ABL1) have poor prognosis. What
about smaller deletions? scFISH permits detection of smaller deletions.
Breakpoint Delineation Using scFISH Probe Clusters
One possible strategy….
Chromosome A
Probe:
1 2
3
4 5
6
7
8
9
Translocates to
chromosome B
tel
cen
Chromosome break
Probe clusters labeled in:
Scale:
First hybridization
~10 kb
Second hybridization
Inferred breakpoint interval:
Third hybridization
.
.
.
Breakpoint Delineation Using scFISH Probe Clusters
Probe:
cen
1
2
3
4
5
6
7
8
9
tel
Probes: 1-9
Pattern:
der(A)
der(B)
B
A
der(B)
der(A)
1-5
B
A
der(A)
der(B)
6-9
B
A
Strategy for Detecting Chromosome 9q34 Deletions by scFISH using
Minimal # of Hybridizations
E S p ro b e
n o t d e le te d
o n d e r (9 )...
No
Conf ir m de le tio n w ith
s c FIS H AS S * a nd B CR^
pr obe s (Aim 2 ) .
Ye s
H yb rid ize w ith 5 ’
AB L * a n d B C R ^
s c FIS H p ro b es ...
5 ’ AB L
in ta c t
5 ’ AB L d e le te d
H yb rid ize w ith
R R P 4 * an d “FIB “^
s c FIS H p ro b es ...
b o th
p ro b es
d e le te d
No de le ti on
pr e s e nt.
Pr o b e s d e n o te d w ith * w ill
b e la b e le d w ith
d ig o x ig e n in ( a n d d e tec te d
w ith a r e d f lu o r oc h r o me ) ,
a n d ^ w ill b e la b e le d w ith
b io tin ( a n d d e te c te d w ith a
g r e e n f lu o r oc h r o me ) af te r
in d ir e c t im mu n o a f f in ity
la b e llin g .
De le tio n b oun da r y
be tw e e n “ FIB” a nd AS S .
“FIB ” in ta c t, R R P 4 d e le te d
H yb rid ize w ith
P R D M1 2 * a n d
3 ’ AB L ^ s c FIS H
p ro b es ...
P R D M1 2
in ta c t
De le tio n b oun da r y be tw e e n
P RDM 1 2 a n d RRP 4 .
P R D M1 2 d e le te d
H yb rid ize w ith
FB P 3 * a n d 3 ’
AB L ^ s c FIS H
p ro b es .
FB P 3 d e le te d
De le tio n b oun da r y be tw e e n
5 ’ FI B a n d 5 ’ FBP 3 .
FB P 3
in ta c t
De le tio n b oun da r y be tw e e n
3 ’ FBP 3 a nd 5 ’ P R DM 1 2
1 to 5 hybridizations necessary to
classify molecular deletion subclass
Cen-ASS-’FIB’-FBP3-PRDM12-RRP4-ABL1-Tel
Identification 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 (>95% homology)
Acute Myelogenous Leukemia (AML M4)
Karyotype: 46,XX,inv(16)(p13q22)
16
Sizes and Locations of Single Copy Intervals in Genes
Detected in Inv(16)(p13q22) AML-Type M4
scFISH 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.
Delineation 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.
Locations of scFISH and Commercial Telomere Probes^
Prediction: >10 % of IMR
patients will have terminal
imbalances with scFISH
probes.
MONOSOMY CHROMOSOME 1P36 SYNDROME
*
*
CDC2L1
Karyotype: 46,XY,del(1)(p36.1).ish del(1)(p36.1)(CDC2L1-)
Chromosome Structure/Organization
• Duplicons, paralogous sequences
• New repetitive sequences
• Chromosomal distribution of single copy
intervals
• Different hybridization efficiency between
homologs (eg. Differential accessibility)
Down Syndrome Critical Region Duplicon Probes
New Repetitive Sequence Observed in
DSCR4 Gene (21q22.3)
DSCR4-1.9 kb
Low stringency wash [4 X SSC]
DSCR4
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.
Why 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.
Chromosome 22: Distribution and Sizes of Single
Copy Intervals
22.0
19.8
17.6
15.4
13.2
Length
11.0
(Kbp)
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
Chromosome 22: Distances between Single Copy
Intervals (>2.3 kb)
Histogram
700
Number of intervals
F re q u e n c y
600
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.
500
400
300
200
Max
Std. Dev = 30657.21
100
Mean = 22332.9
0
0.
0
2 1507.00
10 12 14 16 18 20 22 24 26 N =
20 40 60 80
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 80 0
00 00 00 00
00 00 00 00 00 00 00 00 00 00
0.
0.
0. 0.
.0
.0 .0
.0
.0
.0
.0 .0
.0
.0
0
0
0
0
Distance separating adjacent intervals
V1
Distribution of Distances Between Single Copy Intervals (>2.3 kb):
Nonrandom at Extreme Distances
Normal Q-Q Plot of VAR00002
7
Normal Q-Q Plot of V1
4
3
2
1
0
E x pe c te d N o rm a l
6
-1
-2
untransformed
-3
-4
-100000
0
100000
200000
300000
Observed Value
5
4
3
2
1
2
Observed Value
3
4
5
Log10 Distance
6
7
> 2.3 kb sc intervals separated by
by ~50-1000 bp and by >100kb
more often than expected
from a random distribution.
Future enhancements
• Automation of probe preparation
• Automation of metaphase scanning of
scFISH probes
• Genome-wide single copy (sc) probe map
and design
Automated 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
CMH: Review by
microscopist
Selection of adequate images
Algorithm
and/or
parameter
refinement
Return image coordinates
CMH: Final capture and optimization of individual images
* Automated stage, camera, filter wheel, Z-stack
Summary
• scFISH rapidly generates probes from genomic sequences (40
regions + telomeres; >120 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!
Collaborations/Acknowledgements:
Computational 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