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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. Prenatal, postnatal and neoplastic chromosomal abnormalities

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Probing the Genome with scFISH

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  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

  2. 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.

  3. Molecular Cytogenetic Test: FISH Complementary nucleic acid and chromosomal target DNA bind noncovalently; binding detected by fluorescence.

  4. 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

  5. Availability of Locus Specific Commercial Probes Inherited abnormalities Subtelomeric regions Acquired abnormalities

  6. 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

  7. 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

  8. Nonspecific Hybridization without Cot 1 DNA Blocking

  9. Conventional FISH: Chromosome X Probes Green = DXZ1; Red = KAL1; cosmid clones

  10. 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

  11. 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 Genes GenBank (mRNA) HIRA X7529 6 ZNF74 X71623

  12. 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

  13. 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

  14. Sizes & Locations of Single Copy Intervals in 3 Chromosomal Regions 22q11.2 15q11.2 1p36.3

  15. 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.

  16. 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

  17. 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

  18. 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

  19. PRADER-WILLI and ANGELMAN SYNDROMES * MAGEL2 Karyotype: 46,XY,del(15)(q11.2q13).ish del(15)(q11.2q13)(MAGEL2-)

  20. 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

  21. 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

  22. Localization of scFISH probes on Ensembl reference sequence Complete probe listing with hyperlinks: in Knoll and Rogan, Amer J Med Genetics, in press.

  23. 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)

  24. Chromosome 17p11.2: Smith-Magenis Region Common interstitial deletion involving meiotic mispairing of SMS REP paralogs; Juyal et al, 1996; Potocki et al, 1998

  25. Atypical Deletion in Smith-Magenis Syndrome 17 Deletion* : FLI1 probe Nondeletion:ADORA2B probe

  26. Chromosome 17p11.2: Smith-Magenis Region Our patient: Deleted Intact

  27. 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

  28. *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)

  29. Sizes and Locations of Single Copy Intervals in BCR and ABL1 Genes Chromosome breakage region:

  30. 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

  31. 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.

  32. 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:

  33. 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

  34. 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

  35. 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)

  36. Acute Myelogenous Leukemia (AML M4) Karyotype: 46,XX,inv(16)(p13q22) 16

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

  38. 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.

  39. 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.

  40. Locations of scFISH and Commercial Telomere Probes^ Prediction: >10 % of IMR patients will have terminal imbalances with scFISH probes.

  41. MONOSOMY CHROMOSOME 1P36 SYNDROME * * CDC2L1 Karyotype: 46,XY,del(1)(p36.1).ish del(1)(p36.1)(CDC2L1-)

  42. Chromosome Structure/Organization • Duplicons, paralogous sequences • New repetitive sequences • Chromosomal distribution of single copy intervals • Different hybridization efficiency between homologs (eg. Differential accessibility)

  43. Down Syndrome Critical Region Duplicon Probes

  44. 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.

  45. 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.

  46. 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

  47. 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

  48. 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

  49. Future enhancements • Automation of probe preparation • Automation of metaphase scanning of scFISH probes • Genome-wide single copy (sc) probe map and design

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