Molecular Cytogenetics Clinical Applications of Molecular Expanding the resolution of conventional cytogenetic Cytogenetics analysis • Molecular cytogenetic techniques provide a way • Application of the techniques of Molecular Biology to detect complicated, cryptic and to cytogenetic preparations submicroscopic rearrangements that remain undetected or undecipherable by conventional cytogenetic analysis

FLUORESCENCE IN SITU HYBRIDIZATION HYBRIDIZATION STEPS

Probe with fluorochrome or hapten FISH Generation of Single-Stranded DNA by Denaturation

Application of • FISH is a physical DNA mapping technique in which DNA Probe a DNA probe labeled with a marker molecule is hybridized to chromosomes on a slide, and visualized using a fluorescence microscope

• The marker molecule is either fluorescent itself, or Probe recognizes is detetcted with a fluorescently labeled antibody. Target DNA Sequences Probe hybridizes To Target DNA Sequences

Chromatin Compaction Classification of Chromosomal Sequences

Beta satellite Alpha satellite Classical satellite Telomeric sequences Unique gene sequences Partial chromosome paints

Metaphase chromosome is compacted into a structure that is 50,000 times shorter than its Whole chromosome paints extended length

1 FISH APPLICATIONS FISH TECHNIQUES • Gene Mapping • Chromosome Identification • Metaphase FISH • Aneuploidy Detection • Interphase FISH • Sexing for X-Linked diseases • Marker chromosome Identification • Reverse FISH • Total chromosome Analysis • Multi-color FISH (M-FISH; SKY) • Translocation Analysis • PRINS • Unique Sequence DNA Detection • Fiber FISH • Microdeletion Syndrome Analysis • CGH • Gene Amplification Analysis • DNA Arrays (Chip technology) • Mouse Chromosome Research

GENE MAPPING

Partial Karyotype of Patient

15.3 15.3

15.1 15.1

14 14

13.3 13.3

13.1 13.1 12 12

11.1 11.1

11.2 11.2

12 12

13.1 13.1

13.3 13.3 14 14 Chromosome 5 15 15

21 21

22 22

23.1 23.1

23.3 23.3

31.1 31.1

31.2 31.2

31.3 31.3

32 32

33.1 33.1 33.3 33.3 34 34 Chromosome 11 35.1 35.1

35.3 wcp 11 5 add(5)(q35) 5 add (5)(q35) wcp 5

2 Microdeletion Syndromes Microdeletion Studies Using FISH

Syndrome Chromosome Location Probe/Gene Locus • Deletions of a megabase or so of DNA that are DiGeorge 22q11.2 D22S75 most often too small to be seen under the Velocardiofacial 22q11.2 D22S76 microscope Miller-Dieker 17p13.3 D17S379 • Produce well defined contiguous gene Smith-Magenis 17p11.2 D17S29 syndromes which demonstrate superimposed Prader-Willi 15q11.2 SNRPN features of several different mendelian Angelman 15q11.12 D15S10 diseases(X-linked or autosomal) Williams 7q11.23 Elastin Cri du chat 5p15.2 D5S23 • Defined by high resolution banding or molecular Wolf-Hirschhorn 4p16.3 D4S96 cytogenetic techniques

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Prader-Willi Syndrome Angelman Syndrome

Normal Deleted Chr 15 Chr 15

Prader Willi Probe with control chromosome 15 probe

From Medical Genetics (Jorde, Carey, Bamshad, White; 2nd Ed.)

3 Normal Deleted Chr 7 Chr 7 Deleted Normal Chr 22 Chr 22 Williams Probe with Control Chromosome 7 Probe

Di George Probe with Control Chr 22 Probe

4 FISH on Interphase Nuclei is Interphase FISH vs Metaphase Useful in Specific Clinical Situations FISH

• Prenatal diagnosis aneuploidy screening by • Prenatal diagnosis aneuploidy screening by FISH looks at interphase nuclei derived from chorionic villi or amniocytes FISH looks at interphase nuclei derived from chorionic villi or amniocytes • Preimplantation Genetic Diagnosis aneuploidy screening by FISH looks at interphase blastomere nuclei • Preimplantation Genetic Diagnosis aneuploidy screening by FISH looks at interphase blastomere nuclei

Chromosomes Enumeration by Rapid Prenatal Interphase FISH

From • Trisomies 13, 18 & 21 and Monosomy X are the Metaphase most common aneuploidies related to maternal age or fetal abnormality

• Routine chromosome analysis take 7-10 days To Interphase • Prenatal Interphase FISH provides a rapid way to screen for the common aneuploidies in uncultured amniotic fluid cells in about 1-2 days

Interphase Amniocyte X Y 21

Chromosomes Enumeration Disomy 21 Female Trisomy 21 Male in Chorionic Villi and Amniocytes by Rapid Prenatal Interphase FISH

5 Benefits of Prenatal Interphase FISH Sensitivity of Prenatal Interphase FISH • Trisomies 13, 18 & 21 and Monosomy X are the most common aneuploidies related to maternal age or fetal abnormality Abnormality Sensitivity Specificity PPV NPV

• Routine chromosome analysis take 7-10 days Trisomy 13 98.6% 100% 100% 99.98% Aneuploidy 18 99.5% 100% 100% 99.8% • Prenatal Interphase FISH provides a rapid way to screen for the common aneuploidies in uncultured amniotic fluid cells in about 1-2 Trisomy 21 100% 100% 100% 100% days 45,X 100% 99.98% 98.5% 100% Other Sex 100% 100% 100% 100% ™ Reduces emotional burden on the patient and/or physician in the face of an Chr increased risk for chromosome abnormalities following an abnormal screening All detectable 99.6% 99.98% 99.8% 99.96% result From Tepperberg et al. Prenatal Diagnosis 2001; 21:293-301

¾ Opportunity to reduce anxiety through earlier decision making • Study looked at 5197 pregnancies • Review of Lit includes ~ 30,000 pregnancies

Preimplantation Genetic Preimplantation Genetic Diagnosis Diagnosis • PGD is a very early form of prenatal diagnosis • Method ™ Generate Oocytes/embryos in vitro by ART • Oocytes or embryos obtained in vitro through ™ Biopsy Polar Body/1-2 cells from embryos assisted reproductive techniques are biopsied ™ FISH or PCR for genetic diagnosis ™ Polar bodies for the oocytes • Patients ™ Blastomeres for the embryos ™ Repeated terminations • Only Embryos shown to be free of the disease ™ Moral or religious objections to termination under consideration are subsequently used for ™ Repeated miscarriages due to chromosome abnormality transfer ™ Infertile couples

Biopsy Polar Body Biopsy

• Used by few groups in USA • Polar Body • Need the first and second polar body • Cleavage Stage • Labour intensive • Blastocyst • Only maternal chromosomes examined

From Veeck – Atlas of Embryology

6 Embryo Biopsy – Acid Tyrodes Day 3 - Cleavage Stage

• Used by majority of groups

• Biopsy at 6-10 cell stage

Used by majority of groups • Blastomeres totipotent

• 1-2 cells for analysis

From Veeck – Atlas of Embryology

Courtesy of Kathleen Miller, RMA of NJ

Aneuploidy Analysis of Embryos for Embryo Biopsy Chromosomes 13, 16, 18, 21 & 22

Chr. 18

Chr. 13

Chr. 16

Chr. 21

Chr. 22 Trisomy 22 Courtesy of Kathleen Miller, RMA of NJ

Multiple Hybridizations Add Diagnostic Power Mosaicism Post-Zygotic Non–Disjucntion

Chromosomes 15 17 X

Chr. 13 Chr. 18 Chr. X Chr. 21 Chr. Y Chr. 13 Chr. 16 Chr. 18 Chr. 21 Chr. 22

7 Mosaicism Mosaicism Post-Zygotic Non–Disjucntion Post-Zygotic Non–Disjucntion

Chromosomes Chromosomes 13 13 16 16 18 18 21 21 22 22

Telomeres FISH using Subtelomere Probes

• Highest concentration of genes of any chromosomal region – therefore sub-microscopic deletions and 5p 13q duplications would have a significant impact

• Increased genetic recombination at telomeres

™ Male rate higher than female for most chromosomes

™ Telomeres play a critical role in chromosome pairing at meiosis

Pericentric Inversion Pericentric Inversion FISH

Mother5p 5q Proband

8 Spectral KarYotyping (18q- ; X+)

(Schrök et al – Hum Genet 1997)

Multiple Hybridizations Add D Diagnostic Power N A

C H I P

TECHNOLOGY

Chr. 13 Chr. 18 Chr. X Chr. 21 Chr. Y Chr. 13 Chr. 16 Chr. 18 Chr. 21 Chr. 22

Detection of Partial Aneuploidies – An expensive FISHing Expedition

• Unbalanced rearrangements • Marker chromosomes Marker • Cryptic translocations • Cryptic deletions • Microdeletions

The Need for New Technologies

9 REVERSE FISH FOLLOWING MARKER Spectral KarYotyping CHROMOSOME MICRODISSECTION (18q- ; X+)

18p 18p

(Schrök et al – Hum Genet 1997)

DNA Microarrays D N A

C H I P

TECHNOLOGY

COMPARATIVE GENOMIC HYBRIDIZATION CGH

C G H • In situ hybridization of differentially labelled specimen DNA & normal reference DNA to normal human • Identifies chromosomal gains and losses in a metaphase chromosome spreads. single hybridization procedure

• Effectively reveals any DNA sequence copy • Specimen & reference DNA can be distinguished by number changes (i.e., gains, amplifications, losses their different fluorescent colors. and deletions) in a particular specimen and maps these changes on normal chromosomes

10 DAPI FITC

36.3

36.2

36.1

35

34.3 34.2 34.1

33

32

31

22 LOSS 21

13

12 11 Heterochromatic 11 Region 12

21 22 Texas 23

24

25 Red

31 GAIN

32

41

42

43 44 FITC 0.5 0.75 1.0 1.25 1.5 + Chromosome 1 Texas Red

OVERVIEW OF CGH

The major steps in CGH involve: X Chromosome DAPI FITC • Preparation of normal metaphase spreads Inverted DAPI Chromosome 9

X Chromosome Isolation of high molecular weight DNA from Y Chromosome • specimen (test) and reference (normal) samples.

Chromosome 9 • Labelling of specimen & reference DNA with different color fluorochromes

Texas FITC Red + Y Chromosome Texas Red

OVERVIEW OF CGH (cont)

• In situ hybridization of the labelled specimen & reference DNAs to normal metaphase spreads

• Washing off unbound DNA

• Counterstaining metaphase spreads with DAPI

• Fluorescent microscopy to visualize & capture color ratio differences along the chromosomes

11 CGHCGH MicroarrayMicroarray Methodology Methodology

Control genomic Test genomic DNA DNA

Arrayed clones Automated analysis

Cloned human DNA (BAC/PAC)

CGH Microarray Methodology CGH Microarray Methodology

Trisomy 21 (47,XY,+21) Trisomy 18 (47,XY,+18)

CGH Microarray Methodology Interpreting a Normal CGH Result in a Karyotypically Normal Individual with Clinical Abnormalities

• A normal CGH result has to be interpreted within the boundaries of the test’s limitations

• A normal CGH result does NOT rule out balanced cryptic rearrangements

• A normal CGH result does NOT rule out sub- microscopic imbalances such as microdeletions

12 Benefits of CGH Analysis in Clinical Genetics CGH IN CLINICAL CYTOGENETICS

• Precise identification of extra or missing material The ability of CGH to define more precisely the – Important for diagnostic and prognostic value chromosomal material comprising marker – Important for identifying those genes causative of the clinical phenotype chromosomes and unbalanced translocations may help to further define critical chromosomal • Single step global genome scan prevents regions which are associated with normal and FISHing expedition adverse phenotypic outcomes and thus provide prognostic information for genetic • DNA based analysis counseling. – Quality of metaphase spreads is not a consideration – Non-viable tissues are amenable to analysis

Benefits of CGH Analysis in Clinical Genetics Benefits of CGH Analysis in Clinical Genetics

It is therefore prudent for investigators who are Information derived from such a database utilizing the newer molecular cytogenetic would directly benefit prenatally ascertained techniques, such as reverse FISH and CGH, to cases of chromosomal imbalance, providing report their findings in conjunction with the couples with a means to make rational and clinical presentation so that a comprehensive informed decisions concerning the pregnancy. database can be constructed. In pediatric cases, such information may provide the parents with a realistic prognosis and be important for the clinical management of the infant.

Peripheral Blood Cultures FISH Studies

• Complete Media - Microcultures • All FISH requests require routine chromosome – Whole blood NOT buffy coat analysis – Only need 2 - 3 cc of blood – Turn around time = 72hrs (routine culture) + • Collection tube must contain Sodium Heparin hybridization/FISH analysis time (green top tube). BE SURE TO INVERT TUBE TO PREVENT – Final report issued when all studies completed CLOTTING. • Probe selection must be focused !! • Short term culture takes 72hrs to complete. – Dependent on clinical indication SPECIALIZED ADDITIONAL STUDIES MUST BE NOTED AT THE ONSET TO ENSURE THAT THE CULTURE IS SET UP – Use of multiple probes = $$$$$$$$ & Time APPROPRIATELY.

13 FISH Studies (Cont)

• Probe availablity should be cleared with the laboratory before specimen is collected – Limited stock kept in lab (Shelf life) – Vendor QA • Availability may cause time delay – Research probes have limited availability • Publication does not = immediate clinical application

14