an introduction to Cytogenetics

We are just in the beginning !

The Principles of Clinical Cytogenetics should be divided into sections:

FIRST An overview on the historical perspective Explanation of the concepts involved Description of cytogenetic nomenclature Examples of its use.

SECOND

An overview of the processes involved to provide a fundamental understanding of the labor-intensive nature of chromosome analysis

THIRD

the main focus SHOULD BE ON the various applications of chromosome analysis in clinical settings and the significance of abnormal results

Cytogenetics techniques are:

Confusing C Complex C Concise; Muck background and training C Capable; the role is increasing (a new era !) C

Morphology Histology Histopathology Cytology Special staining Using markers (FC, IHC) Cytogenetics Molecular Genetics

Histochemical staining

M-I-C
Morphology Immunophenotyping Cytogenetics

Cytomorphology

Cytogenetic and Molecular genetic

Immunophenotyping

Micro gene array the idea is existing since mid-80s

Modern Diagnostics in Cancer Management: A multi-modal Approach

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Articles in the Medline database with reference to flow cytometry

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1974: first article

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Increasing reference to (Flow Cytometry) in the medical literature over the past 3 decades
(Alice Longobardi Givan. Flow Cytometry First principles, 2nd edition. Preface article, 2001, A John Wiley & Sons Inc., Publication)

The first impedance based flow cytometry to count cells using 1953 the coulter principle (by Wallace A Coulter, USA). The first fluorescence-based flow cytometer was developed (Wolfgang Ghde, University of Munster, Germany).

1968

Remarkable change on the size of the flow cytometer and 2000s software capability, and up to 17 color could be used at a time.

Just in the last decade the great discoveries of the cell, the nucleus, the chromosome, and DNA have been initiated
An emphasis placed by some on determining the correct number of chromosomes in humans. 1912, von Winiwarter concluded that men have 47 chromosomes and women have 48 !!! 1923, Painter studied (meiotic) chromosomes derived from the testicles Texas State Insane Asylum He definitively reported the human diploid chromosome number to be 48 (double the 24 bivalents he saw) Two years earlier, he had preliminarily reported that some of his better samples produced a diploid number of 46. He also proposed the X and Y sex chromosome mechanism in man. One year later, Levitsky formulated the term karyotype to refer to the ordered arrangement of chromosomes.

The references for the previous 2 works: Painter, T.S. (1923) Studies in mammalian spermatogenesis. II. The spermatogenesis of man. J. Exp. Zool. 37, 291-336

Levitsky G.A. (1924) Materielle Grundlagen der Vererbung. Staatsverlag, Kiew

Jrme Lejeune receives a Joseph P. Kennedy, Jr. Foundation International Award Down syndrome results from an extra chromosome December, 1962

The study of human chromosomes plays a role in 1) Diagnosis 2) Prognosis 3) Monitoring of treatment
The involving conditions seen not only by medical geneticists and genetic counselors, but also by: a) Pediatricians b) obstetrician/gynecologists c) Perinatologists d) Hematologists e) Oncologists f) Endocrinologists g) Pathologists h) Urologists i) Internists j) Family practice physicians.

Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell, especially the chromosomes. It includes: 1) Routine analysis of G-Banded chromosomes 2) Other cytogenetic banding techniques 3) Molecular cytogenetics such (FISH and CGH)

Since its first application to the study of cancer, cytogenetics has taken us:
FROM a state of virtually no knowledge of the chromosome changes in human cancer TO a point at which a staggering body of information is available The latter is evidenced by the nearly 55,000 Cancer karyotypes now included in: (Mitelman Database of Chromosome Aberrations in Cancer) http://cgap.nci.nih.gov/Chromosomes/Mitelman

American College of Medical Genetics

International Standing Committee on Human Cytogenetic Nomenclature ISCN

Cytogenetic testing is often an issue for hospital laboratory personnel and managed care organizations, yet cytogenetics is often less well understood than most specialized laboratory testing.

Why?

Cytogenetic testing is often an issue for hospital laboratory personnel and managed care organizations, yet cytogenetics is often less well understood than most specialized laboratory testing.

Why?

Cytogenetic testing is often an issue for hospital laboratory personnel and managed care organizations, yet cytogenetics is often less well understood than most specialized laboratory testing.

Why?
One can attribute this to several causes: The cytogenetics lab is essentially the only setting in which living cells are required for traditional testing. This unusual sample requirement is a potential source of confusion. Cytogenetics is still perceived, and rightly so, to be as much art as it is science in an era when most clinical testing is becoming more and more automated or high tech. Genetics in general still does not receive sufficient emphasis in the training of medical personnel.

Karl Wilhelm von Ngeli 27th March, 1817-11th May, 1891 a Swiss botanist, studied cell division and pollination

Walther Flemming

1843 - 1905 German biologist and a founder of cytogenetics. He became the director of the Anatomical Institute and stayed there until his death.

The hand drawing of Flemming: Illustrations of cells with chromosomes and mitosis From the book Zellsubstanz, Kern und Zelltheilung

Drawing of a salivary gland cell as published by Flemming. One of over 100 drawings from Flemming's book Zellsubstanz, Kern und Zelltheilung 1885

He was first that:

Studied cell division and chromosomes in details and Published the results in his book (1881-2)

A great step forward in cell biology, occurred? The process of cell division studied in great detail, With the help of powerful new microscopes and dye staining techniques.

Carnegie Institute for science

Washington DC, USA
A joined project between:

Astronomy

(explore the universe)

13 13

Biology

(explore the world around you)

Chemistry

(explore the world under the microscopic)

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12 10 13 13 13

Earth Science

(explore the earth under your feet)

Evolution

(explore the past)

Genetics

(explore what makes you, you)

Medicine

(explore development in health)

Physics

(explore how stuff works)

The great discoveries in Biology -13-

1) Microorganisms (1674( Microscope lens grinder Anton Van Leeuwenhoek accidentally discovers microorganisms in a drop of water. Using his own microscopes, he observes sperm, bacteria and red blood cells. His observations lay the foundation for the sciences of bacteriology and microbiology.

The great discoveries in Biology -132) The Cell Nucleus (1831) While studying an orchid family, botanist Robert Brown identifies the detailed structure within the cells and recognized the nucleus inside the cell.

orchid family is a diverse and widespread family of flowering plants with colorful and fragrant blooms .

The great discoveries in Biology -133) Archaea (1977) Carl Woese an American microbiologist and physicist, famous for defining the Archaea (a new kingdom of life) in 1977. He discovers bacteria are not the only simple-celled prokaryotes on Earth. His three-domain system, based on genetic relationships rather than obvious morphologic similarities, divided life into 23 main divisions, incorporated within three domains: Bacteria, Archaea, and Eucarya.

The great discoveries in Biology -134) Cell Division (1879) Walther Flemming carefully observes that animal cells divide in stages mitosis Eduard Strasburger independently identifies a similar process of cellular division 5) Sex Cells (1884) August Weismann identifies that sex cells must have divided differently to end up with only half of a chromosomal set. This very special division of sex cells is called meiosis. 6) Cell Differentiation (late 19th century( Several scientists participate in the discovery of cell differentiation, eventually leading to the isolation of human embryonic stem cells. During differentiation, a cell turns into one of the many cell types. Cells that are not yet differentiated and have the potential to become any type of cell are called stem cells.

The great discoveries in Genetics -131) Rules of Heredity (1850s(

Gregor Mendel (Austrian monk and botanist) discovers how genetic information is passed down through generations. Mendel's findings are ridiculed during his lifetime and he dies never knowing that he would come to be known as the "father of genetics".

2) Genes Are Located on Chromosomes (1910 1920s)

Thomas Hunt Morgan discovers that genes are located on chromosomes.

3) Genes Control Biochemical Events (1930(

George Beadle and Edward Tatum discover that genes are responsible for the production of enzymes. Their report is the genesis of the "one gene-one enzyme" concept.

The great discoveries in Genetics -13-

4) Some Genes Can Jump (1940(

Barbara McClintock discovers transposons (genes that can jump on a chromosome) while seeking to explain color variations in corn. Transposons are segments of DNA that can move to different positions in the genome of a single cell. These mobile segments of DNA are sometimes called "jumping genes".

5) DNA Is the Genetic Material (1928, 1944, 1952(

Several scientists prove that DNA is the chemical basis of genetic information. Oswald Avery proves that DNA carries genetic information. Linus Pauling discovers that many proteins take the shape of a spiral, like a spring coil. Finally, biochemist Erwin Chargaff finds the arrangement of certain nitrogen bases in DNA always occurs in a 1-to-1 ratio, forming base pairs.

6) DNA Is a Double Helix (1953(

James Watson and Francis Crick describe the DNA molecule. The scientists suggest that the DNA molecule is made of two chains of nucleotides, each in a helix, one going up and the other going down. They show that each strand of the DNA molecule is a template for the other, and that DNA can reproduce itself without changing its structure, except for occasional errors or mutations.

The great discoveries in Genetics -137) .ing the Genetic Code (1960s(
Marshall Nirenberg leads the team that discovers the genetic code, showing that a sequence of three nucleotide bases (a codon) determines each of the 20 amino acids.

8) RNA Conveys Genetic Information (1960s(

A number of scientists discover RNA, a chemical found in the nucleus and cytoplasm of cells with a structure similar to DNA. They find that RNA plays an important role in protein synthesis and other chemical activities in the cell.

9) Restriction Enzymes (1950s 1960s)

Several scientists discover restriction enzymes, biological scissors that recognize and cut specific DNA sequences.

10) RNA Splicing (1976(

Several groups of scientists discover RNA splicing. They learn that for cells to produce protein, DNA is first transcribed into pre-messenger RNA. For reasons that remain unclear, pre-messenger RNA molecules are then spliced to create mature messenger RNA. In many genetic diseases, gene mutations cause errors in the RNA splicing process. Improperly spliced messenger RNA molecules create altered proteins and result in disease.

The great discoveries in Genetics -137) .ing the Genetic Code (1960s(
Marshall Nirenberg leads the team that discovers the genetic code, showing that a sequence of three nucleotide bases (a codon) determines each of the 20 amino acids.

The genetic code is the set of rules by which information encoded in genetic material (DNA or mRNA sequences) is translated into proteins (amino acid sequences) by living cells. The code defines how sequences of three nucleotides, called codons , specify which amino acid will be added next during protein synthesis.

The great discoveries in Genetics -1311) DNA Polymorphism (1985) Alec Jeffreys discovers that some DNA sequences are unique to each individual, leading to the birth of DNA forensics. His DNA technique is first used to hunt down a child molester who killed two girls. The suspect, Colin Pitchfork, is convicted of murder after DNA samples taken from him match semen samples taken from the two dead girls. 12) RNA Interference (1998( Andrew Fire and Craig Mello discover RNA interference (RNAi), in which the presence of small fragments of double-stranded RNA (dsRNA) whose sequence matches a given gene interferes with the expression of that gene. Scientists believe that dsRNAs that trigger RNAi may be usable as drugs .

The great discoveries in Genetics -13-

12. Humans Have 20,000 to 25,000 Genes (20th October, 2004)

Upon sequencing the human genome, it's discovered that humans have approximately 20,000 to 25,000 genes, far fewer than most scientists had predicted. It is hoped that understanding the genome will boost the fields of medicine and biotechnology, eventually leading to cures for diseases such as cancer and Alzheimer's disease.

International Human Genome Sequencing Consortium Describes Finished Human Genome Sequence Researchers Trim Count of Human Genes to 20,000-25,000
The paper appears in the October 21st, ( Nature)

The announcement of the first successful sequencing of the human genome by B. Clinton at the WH on (June 26, 2000), with JC Venter and F Collins was technically premature (only draft sequences were completed).

The first truly complete sequence was published in 2003 in Nature

Why to Study Cytogenetics?

420, 000, 000 IQ

Cytovision GSL-120 system (7.2 version) Flexible Karyotyper Software M-FISH Sofwtare CGH software Two Karyotyoing and FISH review stations Fluorescence microscope with 5-positions filter tubes (DAPI, Green, Red, Blue, Gold, DAPI/Red/Green, Aqua V2)) Leica DM6000B microscope, auto-adjustable Laminar air flow hoods Precision hot plate (regulate the operating temp.) Co2 incubator Hybridization system Water bath, centrifuge, vortex

The technician will

Set up cultures for chromosome analysis on peripheral blood, BM, amniotic
fluid and tissue specimens.

Lean the techniques for harvesting and preparing slides from these various
cultures.

Complete the cytogenetic analysis of test cases and learn how the reporting
procedure is followed in clinical laboratories.

Master the specialized staining techniques required to differentiate special

regions of human chromosomes, also induce specific banding patterns necessary for the identification of genetic changes.

Karyotypes describe:

The number of chromosomes, and what they look like under a LM Attention is paid to their length The position of the centromeres Banding pattern Differences between the sex chromosomes any other physical characteristics. The preparation and study of karyotypes is part of cytogenetics.

Cancer is a genetic disease characterized by DNA changes at either the nucleotide or chromosomal level, or both. At the chromosome level: a) Changes in chromosome numbers (aneuploidy) b) Loss of heterozygosity (LOH), whole or segmental chromosome region loss. c) Chromosomal rearrangements (translocations and inversions) d) Gene duplications or amplifications.

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Progress in understanding the cytogenetic and molecular basis of neoplastic transformation has strengthened the conception of cancer as a genetic disease
Thus, the finding of apparently normal karyotypes in abnormal cells (as is seen in leukemias and various solid tumors) presents an enigma. It can be assumed that cryptic genetic changes are involved in such cases, as has been shown in some tumors and leukemias. These cryptic changes are not discernible with routine cytogenetic methods, but can be studied with special FISH methods: - Spectral karyotyping (SKY) - Multiprobe FISH (M-FISH) - If a specific karyotypic change is suspected, with appropriate molecular means.

Next session Cancer Cytogenetics

karyotyping