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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

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Probing the Genome with scFISH

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Probing the genome with scfish

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

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.


Probing the genome with scfish

Molecular Cytogenetic Test: FISH

Complementary nucleic acid and chromosomal target DNA bind noncovalently; binding detected by fluorescence.


Applications of fish

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


Probing the genome with scfish

Availability of Locus Specific Commercial Probes

Inherited abnormalities

Subtelomeric regions

Acquired abnormalities


Commercial probes properties

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


Probing the genome with scfish

Conventional 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


Probing the genome with scfish

Nonspecific Hybridization without Cot 1 DNA Blocking


Probing the genome with scfish

Conventional FISH: Chromosome X Probes

Green = DXZ1; Red = KAL1; cosmid clones


Sequence based scfish probes properties

OVERCOMES LIMITATIONS OF COMMERCIAL PROBES

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

*US and International patents pending


Probing the genome with scfish

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

ZNF74

GenesGenBank (mRNA)

HIRA X75296

ZNF74 X71623


Step 2 deduce locations of single copy intervals

Step 2: Deduce locations of single copy intervals

  • Computer program compares genomic sequence (>100 kb) withdatabase 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

Step 3: Amplify and purify single copy sequences

  • Sortsequence 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.

Iterate to maximize:

product length,

annealing temperature,

GC% content based on composition

1 2 3 4 kb


Probing the genome with scfish

Sizes & Locations of Single Copy Intervals in 3 Chromosomal Regions

22q11.2

15q11.2

1p36.3


Probing the genome with scfish

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

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


Probing the genome with scfish

Gain or loss of individual genes can be examined due to the high-density and small size of scFISH probes.

Phenotype-Genotype Relationships

Examples:

- Detection of small IC deletions in Angelman and Prader-Willi syndromes

- Detection of atypical deletions in Smith-Magenis syndrome


Probing the genome with scfish

ANGELMAN 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:PWSAS

Deletion~70%~70% Uniparental disomy~25%~5%

Other ~5%~25%

PWS


Probing the genome with scfish

PRADER-WILLI and ANGELMAN SYNDROMES

*

MAGEL2

Karyotype: 46,XY,del(15)(q11.2q13).ish del(15)(q11.2q13)(MAGEL2-)


Probing the genome with scfish

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


Probing the genome with scfish

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 PCR-based DNA methylation analysis.

Probes: PWS-SRO,MAGEL2


Probing the genome with scfish

Localization of scFISH probes on Ensembl reference sequence

Complete probe listing with hyperlinks: in Knoll and Rogan, Amer J Med Genetics, in press.


Probing the genome with scfish

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)


Probing the genome with scfish

Chromosome 17p11.2: Smith-Magenis Region

Common interstitial deletion involving meiotic mispairing of SMS REP paralogs; Juyal et al, 1996; Potocki et al, 1998


Probing the genome with scfish

Atypical Deletion in Smith-Magenis Syndrome

17

Deletion* : FLI1 probe

Nondeletion:ADORA2B probe


Probing the genome with scfish

Chromosome 17p11.2: Smith-Magenis Region

Our patient:

Deleted

Intact


Probing the genome with scfish

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


Probing the genome with scfish

*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).

Chronic Myelogenous Leukemia (CML)

9

22

Karyotype: 46,XX,t(9;22)(q34;q11)


Probing the genome with scfish

Sizes and Locations of Single Copy Intervals in BCR and ABL1 Genes

Chromosome breakage region:


Probing the genome with scfish

Chronic Myelogenous Leukemia and t(9;22)(q34q11.2)

der(22)

9

ABL1, 3-probe cocktail:

IVS3, IVS4-6, IVS11

ABL1, 5-probe cocktail:

Ex1b, IVS1b IVS3, IVS4-6, IVS11

der 22

normal 9

der 9 der 22

normal 9 normal 9


Probing the genome with scfish

ASS

FBP3

PRDM12

RRPR4

ABL

Single 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.


Probing the genome with scfish

Breakpoint 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:


Probing the genome with scfish

Breakpoint Delineation Using scFISH Probe Clusters

1

2 3 4 5

6 7 8 9

Probe:

cen

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


Probing the genome with scfish

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


Probing the genome with scfish

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)


Probing the genome with scfish

Acute Myelogenous Leukemia (AML M4)

Karyotype: 46,XX,inv(16)(p13q22)

16


Probing the genome with scfish

Sizes and Locations of Single Copy Intervals in Genes Detected in Inv(16)(p13q22) AML-Type M4


Probing the genome with scfish

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.


Probing the genome with scfish

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.


Probing the genome with scfish

Locations of scFISH and Commercial Telomere Probes^

Prediction: >10 % of IMR patients will have terminal imbalances with scFISH probes.


Probing the genome with scfish

MONOSOMY CHROMOSOME 1P36 SYNDROME

*

*

CDC2L1

Karyotype: 46,XY,del(1)(p36.1).ish del(1)(p36.1)(CDC2L1-)


Chromosome structure organization

Chromosome Structure/Organization

  • Duplicons, paralogous sequences

  • New repetitive sequences

  • Chromosomal distribution of single copy intervals

  • Different hybridization efficiency between homologs (eg. Differential accessibility)


Probing the genome with scfish

Down Syndrome Critical Region Duplicon Probes


Probing the genome with scfish

New 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.


Probing the genome with scfish

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.


Probing the genome with scfish

Chromosome 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


Chromosome 22 distances between single copy intervals 2 3 kb

Chromosome 22: Distances between Single Copy Intervals (>2.3 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


Distribution of distances between single copy intervals 2 3 kb nonrandom at extreme distances

Distribution of Distances Between Single Copy Intervals (>2.3 kb): Nonrandom at Extreme Distances

untransformed

> 2.3 kb sc intervals separated by

by ~50-1000 bp and by >100kb

more often than expected

from a random distribution.

Log10 Distance


Future enhancements

Future enhancements

  • Automation of probe preparation

  • Automation of metaphase scanning of scFISH probes

  • Genome-wide single copy (sc) probe map and design


Probing the genome with scfish

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 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


Summary

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!


Probing the genome with scfish

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


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