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