Probing the Genome with scFISH
Sequence-based In Situ Detection of Chromosomal Abnormalities at
High Resolution -
Joan HM Knoll, PhD, FACMG, FCCMG
University of Missouri-Kansas City School of Medicine
The Paradigm•Prenatal, postnatal and neoplastic chromosomal abnormalitiesare 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 diagnostictechnologies that stratify patients into risk categories definedby 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 abnormalitiesSubtelomeric regionsAcquired 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 copy gene sequences
repetitive sequences
Labeled and denatured probe DNA:Excess of Denatured Competitor DNA: (Cot 1 DNA)
+
Preannealing
single stranded DNA
double stranded DNA
Hybridization (repetitive sequences are disabled)
Chromosome DNA on microscope slide
Probe
Detection by fluorescence
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
Chromosome 22genomic sequence
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.
HIRA
ZNF74
Example:DiGeorge, Shprintzen,
Velocardiofacial Syndromes
OMIM No. 188400
Genes GenBank (mRNA)
HIRA X75296
ZNF74 X71623
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 kbIterate 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
*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.
Genomic Interval Length Needed to Develop Probes
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
Examples:
- Detection of small IC deletions in Angelman and Prader-Willi syndromes
- Detection of atypical deletions in Smith-Magenis syndrome
Phenotype-Genotype Relationships
Gain or loss of individual genes can be examined due to the high-density and small size of scFISH probes.
Etiology: PWS AS
Deletion ~70% ~70% Uniparental disomy ~25% ~5% Other ~5% ~25%
• 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
ANGELMAN and PRADER-WILLI SYNDROMES
AS
PWS
PRADER-WILLI and ANGELMAN SYNDROMES
Karyotype: 46,XY,del(15)(q11.2q13).ish del(15)(q11.2q13)(MAGEL2-)
MAGEL2
*
CHROMOSOME 15q11.2q13: AS/PWS REGION
Nicholls et al, 1989 Knoll et al, 1989 Gregory et al, 1990Saitoh et al, 1996
Common deletion
PWS IC deletion (SRO)
Detection of the PWS Imprinting Center by scFISH
Probes: PWS-SRO, MAGEL2
scFISH/FISH* detection rate:
PWS: ~99% of abnormalities
AS: ~80% of abnormalities (not UBE3A mutations)
scFISH IC probes potentially offer an alternative to PCR-based DNA methylation analysis.
*includes replication timing FISH assay for UPD (White et al. 1996).
Chromosome/Disorder
Gene Interval Cytogenetic nomenclature
15/Prader-Willi,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/AML-M4
PLA2G10PKDPM5
IVS 3IVS 12-Exon 15~100 kb upstream
ish inv(16)(p13q22)(PLA2G10 mv, PKD mv, PM5 sp)
Localization of scFISH probes on Ensembl reference sequence
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
Deletion* : FLI1 probe Nondeletion: ADORA2B probe
17
Chromosome 17p11.2: Smith-Magenis Region
Our patient:Deleted
Intact
•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
Delineation of Translocation Breakage/Deletion Intervals : Chronic Myelogeneous Leukemia (CML)
Karyotype: 46,XX,t(9;22)(q34;q11)
Chronic Myelogenous Leukemia (CML)
9 22
*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).
Chromosome breakage region:
Sizes and Locations of Single Copy Intervals in BCR and ABL1 Genes
ABL1, 5-probe cocktail:
Ex1b, IVS1b IVS3, IVS4-6, IVS11
ABL1, 3-probe cocktail:
IVS3, IVS4-6, IVS11
Chronic Myelogenous Leukemia and t(9;22)(q34q11.2)
der(22)
der 9 der 22normal 9 normal 9
der 22 normal 9
9
ASS FBP3 PRDM12 RRPR4 ABL
cen tel
bp
Patients with large deletions (ASS-ABL1) have poor prognosis. What about smaller deletions? scFISH permits detection of smaller deletions.
Single Copy Intervals ( 1500 bp) between the ASS & ABL1 Genes on Chromosome 9q34
Chromosome break
Chromosome A Translocates to chromosome B
cen tel
1 2 3 4 5 6 7 8 9Probe:
Breakpoint Delineation Using scFISH Probe ClustersOne possible strategy….
First hybridization
Second hybridization
Third hybridization...
Probe clusters labeled in:
~10 kb
Scale:
Inferred breakpoint interval:
Probes: 1-9 Pattern: der(B)der(A)
AB
1-5
der(B)der(A)
AB
Breakpoint Delineation Using scFISH Probe Clusters
6-9der(B)der(A)
AB
cen tel
1 2 3 4 5 6 7 8 9Probe:
ES probe not deleted on der (9)...
Hybridize with 5’ ABL* and BCR ̂scFISH probes... 5’ ABL
intact
No deletion present.
Yes
No
Hybridize with RRP4* and “FIB“^ scFISH probes...
5’ ABL deleted
Deletion boundary between “FIB” and ASS.
Confirm deletion with scFISH ASS* and BCR^
probes (Aim 2).
both probes deleted
Hybridize with PRDM12* and 3’ ABL ̂scFISH
probes...
“FIB” intact, RRP4 deleted
Deletion boundary between PRDM12 and RRP4. PRDM12
intact
Hybridize with FBP3* and 3’ ABL^ scFISH
probes.
Deletion boundary between 3’ FBP3 and 5’ PRDM12 FBP3
intact
PRDM12 deleted
Deletion boundary between 5’ FIB and 5’ FBP3.
Probes denoted with * w ill be labeled w ith digoxigenin (and detected with a red f luorochrome), and ^ w ill be labeled w ith biotin (and detected with a green f luorochrome) after indirect immunoaffinity labelling.
FBP3 deleted
Strategy 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
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)
Karyotype: 46,XX,inv(16)(p13q22)
16
Acute Myelogenous Leukemia (AML M4)
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)
normal inv(16)(p13q22)*
cell 1
cell 2
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 havesubtelomeric 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.
Prediction: >10 % of IMR patients will have terminal imbalances with scFISH probes.
Locations of scFISH and Commercial Telomere Probes^
*
MONOSOMY CHROMOSOME 1P36 SYNDROME
Karyotype: 46,XY,del(1)(p36.1).ish del(1)(p36.1)(CDC2L1-)
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
Low stringency wash [4 X SSC] High stringency wash [1 X SSC]
DSCR4-1.9 kb DSCR4
New Repetitive Sequence Observed in DSCR4 Gene (21q22.3)
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
19.8
17.6
15.4
13.2
11.0
8.8
6.6
4.4
2.2
0.0
22.0
Length(Kbp)
Centromere Telomere
Chromosomal coordinate (Mbp)0.0 3.4 6.8 10.2 13.6 17.0 20.4 23.8 27.2 30.6 34.0
Chromosome 22: Distances between Single Copy Intervals (>2.3 kb)
V1
HistogramF
req
uenc
y700
600
500
400
300
200
100
0
Std. Dev = 30657.21
Mean = 22332.9
N = 1507.00
Nu
mb
er o
f in
terv
als
Max
Distance separating adjacent intervals
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.
Distribution of Distances Between Single Copy Intervals (>2.3 kb): Nonrandom at Extreme Distances
Normal Q-Q Plot of VAR00002
Observed Value
7654321
Exp
ect
ed N
orm
al V
alue
7
6
5
4
3
2
Log10 Distance
Normal Q-Q Plot of V1
Observed Value
3000002000001000000-100000
Exp
ecte
d N
orm
al
4
3
2
1
0
-1
-2
-3
-4
untransformed
> 2.3 kb sc intervals separated by by ~50-1000 bp and by >100kb more often than expectedfrom 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 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 parametermicroscopist refinement
Selection of adequate images
Return image coordinates
CMH: Final capture and optimization of individual images
* Automated stage, camera, filter wheel, Z-stack
Automated slide processing schema
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