During the Metaphase type of cell division in the Bone Marrow, Karyotyping method gives the visual representation of the 46 chromosomes paired and arranged in decreasing order of size. This representation is useful in leukemia purposes. This method is a difficult one because these chromosomes appear distorted, overlapped, and their images are usually blurred with undefined edges. So here, Karyotyping uses new mutual information method which is proposed to increase the discriminate power of the G-banding pattern dissimilarity between chromosomes and improve the performance of the classifier. This algorithm is formulated as such a method of combinatorial optimization problem. Where the distances between homologous chromosomes are minimized and the distances between non homologous ones are maximized. It is solved by using an integer programming approach. In this project chromosome dataset Lisbon-K1 (LK1) chromosome dataset with 9200 chromosomes was used for this study.


Introduction 1.1 The study of chromosome morphology and its relation with some genetic diseases is the main goal of cytogenetic. Normal human cells have 23 classes of large linear nuclear chromosomes, in a total of 46 chromosomes per cell. The chromosome contains approximately 30 000 genes (genotype) and large tracts of non coding sequences. The analysis of genetic material can involve the examination of specific chromosomal regions using DNA probes, e.g., fluorescent in situ hybridization (FISH) called molecular cytogenetic, comparative Genomic hybridization (CGH) , or the morphological and pattern analysis of entire chromosomes, the conventional cytogenetic, which is the focus of this paper. These cytogenetic studies are very important in the detection of acquired chromosomal abnormalities, such as translocations, duplications, inversions, deletions, monosomies, or trisomies. These techniques are particularly useful in the diagnosis of cancerous diseases and are the preferred ones in the characterization of the different types of leukemia, which is the motivation of this paper . The pairing of chromosomes is one of the main steps in conventional cytogenetic analysis where a correctly ordered karyogram is produced for diagnosis of genetic diseases based on the patient karyotype. The karyogram is an image representation of the stained human chromosomes with the widely used Giemsa Stain metaphase spread (G-banding) , where the chromosomes are arranged in 22 pairs of somatic homologous elements plus two sex-determinative chromosomes (XX for the female or XY for the male), displayed in decreasing order of size. A karyotype is the set of characteristics

extracted from the karyogram that may be used to detect chromosomal abnormalities. The metaphase is the step of the cellular division process where the chromosomes are in their most condensed state. This is the most appropriated moment to its visualization and abnormality recognition because the chromosomes appear well defined and clear. The pairing and karyotyping procedure, usually done manually by visual inspection, is time consuming and technically demanding. The application of the G-banding procedure to the chromosomes generates a distinct transverse banding pattern characteristic for each class, which is the most important feature for chromosome classification and pairing. The International System for Cytogenetic Nomenclature (ISCN) provides standard diagrams/ideograms of band profiles, as for all the chromosomes of a normal human, and the clinical staff is trained to pair and interpret each specific karyogram according to the ISCN information. Other features, related to the chromosome dimensions and shape, are also used to increase the discriminative power of the manual or automatic classifiers. 1.2 CHROMOSOME A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes,regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions. Chromosomes vary widely between different organisms. The DNA molecule may be circular or linear, and can be composed of 100,000 to 10,000,000,000[1] nucleotides in a long chain. Typically, eukaryotic cells (cells with nuclei) have large linear chromosomes andprokaryotic cells (cells without

The structure of chromosomes and chromatin varies through the cell cycle. although there are many exceptions to this rule. However. and passed successfully to their daughter cells so as to ensure the genetic diversity and survival of their progeny. mitochondria in most eukaryotes and chloroplasts in plants have their own small chromosomes. circular DNA molecules called plasmids. Chromosomes may exist as either duplicated or unduplicated. nuclear chromosomes are packaged by proteins into a condensed structure called chromatin. a large body of work uses the term chromosome regardless of chromatin content. If these structures are manipulated incorrectly. or it may unexpectedly evadeapoptosis leading to the progression of cancer. sometimes accompanied by one or more smaller. In prokaryotes and viruses.defined nuclei) have smaller circular chromosomes. whereas duplicated chromosomes contain two identical copies (called chromatids) joined by a centromere. the cell may undergo mitotic catastrophe and die. for example. divided. This allows the very long DNA molecules to fit into the cell nucleus. Chromosomal recombination plays a vital role in genetic diversity. Compaction of the duplicated chromosomes during mitosis and meiosis results in the classic four-arm structure (pictured to the right). the term genophore is more appropriate when no chromatin is present. Also. These small circular genomes . In practice "chromosome" is a rather loosely defined term. cells may contain more than one type of chromosome. In prokaryotes. Chromosomes are the essential unit for cellular division and must be replicated. which is tightly coiled in on itself. DNA is usually arranged as a circle. through processes known as chromosomal instability and translocation. In eukaryotes. Unduplicated chromosomes are single linear strands.

The individual would have Down Syndrome and his/her karyotype would be written 47. An example of aneuploidy is trisomy 21.g. reflecting their bacterial origins. members of the same species have the same numbers of types of chromosomes (with the exception of sex chromosomes in males and females if sex is chromosomally determined).+21. Such individuals are called euploid and have the wild-type chromosome complement for the species.XY or 47. . rather than 2.3 MUTATIONS IN CHROMOSOME NUMBER Normally. XX (female) or 46 XY (male). Euploid human karyotypes are 46.are also found in mitochondria and chloroplasts. a extra copy of human chromosome 21).+21. the individual carrying the mutation is said to be aneuploid. Chromosomal Mutations are substantial changes in chromosome structure that are large enough to be visible by karyotyping (see lab manual) and thus typically affect more than one gene. copies of chromosome 21.XX. The simplest gonophores are found in viruses: these DNA or RNA molecules are short linear or circular gonophores that often lack structural proteins. 1. in which an individual has 3. If the mutation involves only one or a few chromosomes in the genome (e.

so that each daughter cell inherits one set of chromatids. Such a failure of the separation of homologous chromosomes or sister chromatids is called nondisjunction. 1. chromosomes are structurally highly condensed. They cease to function as accessible genetic material (transcription stops) and become a compact transportable form. the chromatin strands become more and more condensed.1 chromosome paired model Aneuploidy is usually caused by spindle fiber failure in meiosis I or II. along with special proteins. The shorter arms are called p arms (from the French petit. In spite of their appearance.Fig 1. the chromatids are uncoiled and DNA can again be transcribed.4 Metaphase chromatin and division In the early stages of mitosis or meiosis (cell division). . longer-lasting attachment in this region. small) and the longer arms are called q arms (q follows p in the Latin alphabet. A special DNA base sequence in the region of the kinetochores provides. The microtubules then pull the chromatids apart toward the centrosomes. microtubules grow from centrosomes located at opposite ends of the cell and also attach to the centromere at specialized structures called kinetochores. This compact form makes the individual chromosomes visible. This is the only natural context in which individual chromosomes are visible with an optical microscope. one of which is present on each sister chromatid. and they form the classic four arm structure. During mitosis. Once the cells have divided. a pair of sister chromatids attached to each other at the centromere. q-g "grande"). which enables these giant DNA structures to be contained within a cell nucleus.

organelles and cell membrane into two cells containing roughly equal shares of these cellular components. . in two separate nuclei. which use the bone marrow vasculature as a conduit to the body's systemic circulation.1 MITOSIS Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets. bone marrow accounts for approximately 2. The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day. It is generally followed immediately by cytokinesis.5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones. On average. which divides the nuclei.1. In humans. cytoplasm. bone marrow in large bones produces new blood cells.6 kg (5. producing the lymphocytes that support the body's immune system CHAPTER 2 2.7 lbs). in adults weighing 65 kg (143 lbs). Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle—the division of the mother cell into two daughter cells. bone marrow constitutes 4% of the total body mass of humans. [1] Bone marrow is also a key component of the lymphatic system.

This occurs most notably among the fungi and slime moulds. where chromosomes divide within an intact cell nucleus. The cell then divides in cytokinesis. while fungi such as Aspergillus nidulans and Saccharomyces cerevisiae (yeast) undergo a "closed" mitosis. "mitosis" is often used interchangeably with "mitotic phase". However. forming single cells with multiple nuclei. The process of mitosis is fast and highly complex. Mitosis occurs only in eukaryotic cells and the process varies in different species. divide by a process called binary fission.[1] Prokaryotic cells. anaphase and telophase. which lack a nucleus. . These stages are interphase. but is found in various different groups. During mitosis the pairs of chromatids condense and attach to fibers that pull the sister chromatids to opposite sides of the cell. to produce two identical daughter cells which are still diploid cells. prometaphase. This accounts for approximately 10% of the cell cycle. where the nuclear envelope breaks down before the chromosomes separate. prophase. metaphase. Even in animals. there are many cells where mitosis and cytokinesis occur separately. for instance during certain stages of fruit fly embryonic development. animals undergo an "open" mitosis. The sequence of events is divided into stages corresponding to the completion of one set of activities and the start of the next.genetically identical to each other and to their parent cell. cytokinesis and mitosis may occur independently. For example. Errors in mitosis can either kill a cell through apoptosis or cause mutations that may lead to cancer. Because cytokinesis usually occurs in conjunction with mitosis.

Each chromosome now has an identical copy of itself. This occurs during the S phase of interphase. and together the two are called sister chromatids. the period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs. . The sister chromatids are held together by a specialized region of the chromosome known as the centromere. Because each resultant daughter cell should be genetically identical to the parent cell. These two cells are identical and do not differ in any way from the original parent cell.Fig 3 Mitosis cell division The primary result of mitosis is the transferring of the parent cell's genome into two daughter cells. The genome is composed of a number of chromosomes—complexes of tightly-coiled DNA that contain genetic information vital for proper cell function. the parent cell must make a copy of each chromosome before mitosis.

giving rise to two daughter cells. so they are renamed to sister chromosomes. As a matter of convention.the cell begins cytokinesis. the parent cell will be split in half.In most eukaryotes. A new nuclear envelope forms around the separated sister chromosomes. As mitosis completes. As the cell elongates. However. each with a replica of the original genome. In animal cells. the daughter cells will construct a new dividing cell wall between each other. the nuclear envelope which segregates the DNA from the cytoplasm disassembles. corresponding sister chromosomes are pulled toward opposite ends. In plant cells. The chromosomes align themselves in a line spanning the cell. each sister chromatid is now considered a chromosome. because of the non-involvement of nuclear dynamics and lack of linear chromosomes. Eventually. the cell pinches inward where the imaginary line used to be (the area of the cell membrane that pinches to form the two daughter cells is called the cleavage furrow). Microtubules — essentially miniature strings— splay out from opposite ends of the cell and shorten. the process of binary fission is very much different from the process of mitosis. . pulling apart the sister chromatids of each chromosome. Prokaryotic cells undergo a process similar to mitosis called binary fission. separating the two developing nuclei.

the cell grows by producing proteins and cytoplasmic organelles.2 Phases of cell cycle and mitosis Interphase Fig 4 The cell cycle The mitotic phase is a relatively short period of the cell cycle. During all three phases. However.1 Preprophase In plant cells only. a cell grows (G1). and finally it divides (M) before restarting the cycle. the nucleus has to migrate into the center of the cell before mitosis can begin. It alternates with the much longer interphase. Thus. a transverse sheet of cytoplasm that bisects the cell along the future plane of cell . chromosomes are replicated only during the S phase. This is achieved through the formation of a phragmosome.2. The phases follow one another in strict order and there are "checkpoints" that give the cell the cues to proceed from one phase to another. continues to grow as it duplicates its chromosomes (S).2. mainly via proteins. S (synthesis). prophase is preceded by a pre-prophase stage. In highly vacuolated plant cells. 2. grows more and prepares for mitosis (G 2). and G2 (second gap). All these phases in the interphase are highly regulated. where the cell prepares itself for cell division. Interphase is divided into three phases: G1 (first gap).

and microtubules have invaded the nuclear Prophase space. The preprophase band disappears during nuclear envelope disassembly and spindle formation in prometaphase. . instead. after the nuclear membrane breaks down. These microtubules can attach to kinetochores or they can interact with opposing microtubules. Cytokinesis has already begun. The cells of higher plants (such as the flowering plants) lack centrioles. Early anaphase: The Prometaphase: The kinetochore nuclear membrane has microtubules shorten.division. Telophase: The decondensing chromosomes are surrounded by nuclear membranes. The chromosomes have chromatin has condensed. the pinched area is known as the cleavage furrow. microtubules form a spindle on the surface of the nucleus and are then organized into a spindle by the chromosomes themselves. In addition to phragmosome formation. degraded. Prophase: The two round objects above the nucleus Metaphase: The are the centrosomes. aligned at the metaphase plate. preprophase is characterized by the formation of a ring of microtubules and actin filaments (called preprophase band) underneath the plasma membrane around the equatorial plane of the future mitotic spindle. This band marks the position where the cell will eventually divide.

At the onset of prophase. A cell inherits a single centrosome at cell division. Molecular motor proteins then push the centrosomes along these microtubules to opposite sides of the cell. the replicated chromosomes have two sister chromatids. which are made of a pair of centrioles found in most eukaryotic animal cells. Since the genetic material has already been duplicated earlier in S phase. the genetic material in the nucleus is in a loosely bundled coil called chromatin. The centrosome is the coordinating center for the cell's microtubules. chromatin condenses together into a highly ordered structure called a chromosome. which is replicated by the cell with the help of the nucleus before a new mitosis begins. The two centrosomes nucleate microtubules (which may be thought of as cellular ropes or poles) to form the spindle by polymerizing soluble tubulin. they are not essential for the . Chromosomes are typically visible at high magnification through a light microscope. Although centrioles help organize microtubule assembly. giving a pair of centrosomes. Close to the nucleus are structures called centrosomes.Fig 5 Micrograph showing condensed chromosomes in blue and the mitotic spindle in green during prometaphase of mitosis Normally. bound together at the centromere by the cohesin protein complex.

and it occurs in most multicellular organisms. and centrosomes are not always used in mitosis. This is called open mitosis. Although the kinetochore structure and function are not fully understood. Prometaphase is sometimes considered part of prophase. since they are absent from plants. undergo a variation called closed mitosis where the spindle forms inside the nucleus. one attached at each chromatid.2 Prometaphase The nuclear envelope disassembles and microtubules invade the nuclear space. coupled with polymerisation and depolymerisation of microtubules. When the spindle grows to sufficient length. 2.2. using energy from ATP to "crawl" up the tube toward the originating centrosome. the motor activates. When a microtubule connects with the kinetochore. . or its microtubules are able to penetrate an intact nuclear envelope.formation of the spindle. on an average 20 ). it is the point where microtubules attach themselves to the chromosome ( about 1-40 in number. kinetochore microtubules begin searching for kinetochores to attach to. it is known that it contains some form of molecular motor. provides the pulling force necessary to later separate the chromosome's two chromatids. Each chromosome forms two kinetochores at the centromere. A number of nonkinetochore microtubules find and interact with corresponding nonkinetochore microtubules from the opposite centrosome to form the mitotic spindle. such as algae or trichomonads. A kinetochore is a complex protein structure that is analogous to a ring for the microtubule hook. Fungi and some protists. This motor activity.

3 Metaphase A cell in late metaphase. analogous to a tug-of-war between people of equal strength. The centromeres of the chromosomes. In certain types of cells. It is also one of the main phases of mitosis because without it cytokinesis would not be able to occur. convene along the metaphase plate or equatorial plane.In the fishing pole analogy. All chromosomes (blue) but one have arrived at the metaphase plate. The centrosome acts as the "reel" that draws in the spindle fibers or "fishing line". in some sense. . the chromosomes come under longitudinal tension from the two ends of the cell. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. the kinetochore would be the "hook" that catches a sister chromatid or "fish"." Microtubules find and attach to kinetochores in prometaphase. Metaphase comes from the Greek meaning "after. an imaginary line that is equidistant from the two centrosome poles. As a result. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. Then the two centrosomes start pulling the chromosomes through their attached centromeres towards the two ends of the cell. only roughly lining up along the midline. 2.

” “back. 2. Two events then occur: first. the nonkinetochore microtubules elongate. allowing them to separate. while late anaphase is the elongation of the microtubules and the chromosomes being pulled farther apart. it is thought that unattached kinetochores generate a signal to prevent premature progression to anaphase without all chromosomes being aligned. Early anaphase is usually defined as the separation of the sister chromatids. Next. These two stages are sometimes called early and late anaphase. the cell has succeeded in separating identical copies of the genetic material into two distinct populations. . The signal creates the mitotic spindle checkpoint.Because proper chromosome separation requires that every kinetochore be attached to a bundle of microtubules (spindle fibres).” “against.” or “re-”). At the end of anaphase. the cell proceeds to anaphase (from the Greek meaning “up. pulling the centrosomes (and the set of chromosomes to which they are attached) apart to opposite ends of the cell. The force that causes the centrosomes to move towards the ends of the cell is still unknown. which have now become distinct sister chromosomes. These sister chromatids.4 Anaphase When every kinetochore is attached to a cluster of microtubules and the chromosomes have lined up along the metaphase plate. the proteins that bind sister chromatids together are cleaved. are pulled apart by shortening kinetochore microtubules and move toward the respective centrosomes to which they are attached. although there is a theory that suggests that the rapid assembly and breakdown of microtubules may cause this movement.

Mitosis is complete. 2. however.5 Telophase Telophase (from the Greek meaning "end") is a reversal of prophase and prometaphase events.5 Cytokinesis Cilliate undergoing cytokinesis. At telophase.2. pinching off the separated nuclei. using fragments of the parent cell's nuclear membrane. with the cleavage furrow being clearly visible Cytokinesis is often mistakenly thought to be the final part of telophase. a cleavage furrow (pinch) containing a contractile ring develops where the metaphase plate used to be. In animal cells. unfold back into chromatin. Corresponding sister chromosomes attach at opposite ends of the cell. forms around each set of separated sister chromosomes. In both animal and plant cells. the nonkinetochore microtubules continue to lengthen. but cell division is not yet complete. now surrounded by new nuclei. cytokinesis is a separate process that begins at the same time as telophase. but rather a separate process. Cytokinesis is technically not even a phase of mitosis. A new nuclear envelope. cell . necessary for completing cell division. It "cleans up" the after effects of mitosis. elongating the cell even more. Both sets of chromosomes.

e.5. zygote and also the basis of the growth of a multicellular body..3 Cell replacement In some parts of body. cells are constantly sloughed off and replaced by new ones.1Significance Mitosis is important for the maintenance of the chromosomal set. which move along microtubules to the middle of the cell. The end of cytokinesis marks the end of the M-phase.5. Following are the occasions in the lives of organism where mitosis happens: 2. whereas some green algae use a phycoplast microtubule array during cytokinesis.5. The phragmoplast is a microtubule structure typical for higher plants. Each daughter cell has a complete copy of the genome of its parent cell.g.e.division is also driven by vesicles derived from the Golgi apparatus. 2. skin and digestive tract. Similarly. 2. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis.5. New cells are formed by mitosis and so are exact copies of the cells being replaced. separating the two nuclei. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell.4 Regeneration . 2.2 Development and growth The number of cells within an organism increases by mitosis. This is the basis of the development of a multicellular body from a single cell i.

2.Some organisms can regenerate their parts of bodies. the process may go wrong. they fail to complete cell division and retain both nuclei in one cell. The production of new cells is achieved by mitosis. For example. For example. resulting in binucleated cells. The cells at the surface of hydra undergo mitosis and form a mass called bud. One daughter cell will receive both sister chromosomes and the other will receive none.5. a condition known as trisomy. a chromosome may fail to separate during anaphase.5. This results in the former cell having three chromosomes containing the same genes (two sisters and a homologue). In non-disjunction. Occasionally when cells experience nondisjunction.7 Consequences of errors Although errors in mitosis are rare. sea star regenerates its lost arm through mitosis. Mitosis continues in the cells of bud and it grows into a new individual. The same division happens during asexual reproduction or vegetative propagation in plants. .6 Asexual reproduction Some organisms produce genetically similar offspring through asexual reproduction. Mitotic errors can be especially dangerous to the organism because future offspring from this parent cell will carry the same disorder. especially during early cellular divisions in the zygote. the hydra reproduces asexually by budding. a condition known as monosomy. These cells are considered aneuploid. and the latter cell having only one chromosome (the homologous chromosome). 2. a condition often associated with cancer.

non-homologous chromosome. its organelles disintegrate and reform in a matter of hours. This phenomenon is called metastasis or spreading of disease. Malignant tumors are also known as cancerous tumours and their cells are called cancerous tumours. but in reverse orientation.Mitosis is a demanding process for the cell. The effect of these genetic abnormalities depends on the specific nature of the error. When tissues more than the requirement are synthesized in a single organ. causing inversion. which goes through dramatic changes in ultrastructure. Now what happens is that cell abnormally continue to divide at a single place. Such tumours can send cancer cells to other parts in body where new tumours may form. All cells have genes that control the timing and number of mitosis. causing deletion. . it results in the formation of Tumors. causing chromosomal duplication. Occasionally. An arm of the chromosome may be broken and the fragment lost. It results in the synthesis of execessive tissue growths. Errors in the control of mitosis may cause cancer. The fragment may incorrectly reattach to another. chromosomes may become damaged. and chromosomes are jostled constantly by probing microtubules. It may reattach to the original chromosome. It results in abnormal cell growth. it may be treated erroneously as a separate chromosome. Or. As long as these tumours remain in their original location they are called benign tumours. causing translocation. As soon as they start to move and invade other cells there are said to be malignant tumours. Benign tumours are not harmful as soon as they are not moving. sometimes mutuations occur in such genes and cells continue to divide.

This process may also be referred to as endoreduplication and the cells as endoploid. carrying genetic information. resulting in cells with many copies of the same chromosome occupying a single nucleus. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. Metaphase accounts for approximately 4% of the cell cycle's duration. align in the middle of the cell before being separated into each of the two daughter cells. In certain types of cells. analogous to a tug of war between equally strong people. from the ancient Greek(between) and (stage). Preceded by events in prometaphase and followed by anaphase. is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes. Early events of metaphase can . microtubules formed in prophase have already found and attached themselves to kinetochores in metaphase. only roughly lining up along the middleline.7 Metaphase Metaphase. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly.6 Endomitosis Endomitosis is a variant of mitosis without nuclear or cellular division. An example of a cell that goes through endomitosis is the megakaryocyte. 2.2. The centromeres of the chromosomes convene themselves on the metaphase plate (or equatorial plate). an imaginary line that is equidistant from the two centrosome poles.

does the cell enter anaphase. Staining of the slides. Metaphase chromosomes make the classical picture of chromosomes (karyotype). when every kinetochore is properly attached to a bundle of microtubules. even if most of the kinetochores have been attached and most of the chromosomes have been aligned. produces a pattern of in total up to several hundred bands. Normal metaphase spreads are used in . Such a signal creates the mitotic spindle checkpoint. Only after all chromosomes have become aligned at the metaphase plate. 2. as chromosomes with connected kinetochores will start the events of metaphase individually before other chromosomes with unconnected kinetochores that are still lingering in the events of prometaphase. Chromosomes are condensed(Thickened) and highly coiled in metaphase. and separase. This would be accomplished by regulation of the anaphase-promoting complex. cells are grown in short term culture and arrested in metaphase using mitotic inhibitor. It is thought that unattached or improperly attached kinetochores generate a signal to prevent premature progression to anaphase. Further they are used for slide preparation and banding (staining) of chromosomes to be visualised under microscope to study structure and number of chromosomes (karyotype). which makes them most suitable for visual analysis. often with Giemsa (G banding) or Quinacrine. For classical cytogenetic analyses. securin.coincide with the later events of prometaphase.8 Metaphase in cytogenetics and cancer studies The analysis of metaphase chromosomes is one of the main tools of classical cytogenetics and cancer studies. One of the cell cycle checkpoints occurs during prometaphase and metaphase.

Inspection of the stained metaphase chromosomes allows the determination of numerical and structural changes in the tumor cell genome. losses of chromosomal segments or translocations. such as bcr-abl in chronic myelogenous leukemia. which may lead to chimeric oncogenes. . Malignant cells from solid tumors or leukemia samples can also be used for cytogenetic analysis to generate metaphase preparations.methods like FISH and as a hybridization matrix for comparative genomic hybridization (CGH) experiments. for example.

such as. and what they look like under a light microscope. to study chromosomal aberrations. Polyploid cells have multiple copies of chromosomes and haploid cells have single copies. be sex chromosomes. The term is also used for the complete set of chromosomes in a species. ordered by size and position of centromere for chromosomes of the same size.CHAPTER 3 KARYOTYPING A karyotype is the number and appearance of chromosomes in the nucleus of an eukaryotic cell. So. [4] The preparation and study of karyotypes is part of cytogenetics. any differences between the sex chromosomes. autosomal chromosomes are present in two copies. or may not. in humans 2n = 46. and the results may be used in evolutionary biology and medicine. There may. in normal diploid organisms. . banding pattern. Thus. The chromosomes are depicted (by rearranging a microphotograph) in a standard format known as a karyogram or idiogram: in pairs. taxonomic relationships. and to gather information about past evolutionary events. and any other physical characteristics. Karyotypes can be used for many purposes. Karyogram of human male using Giemsa staining. the position of the centromeres. The study of whole sets of chromosomes is sometimes known as karyology. The study of karyotypes is important for cell biology and genetics. Attention is paid to their length. Karyotypes describe the number of chromosomes. cellular function. The basic number of chromosomes in the somatic cells of an individual or a species is called the somatic number and is designated 2n. In the germ-line (the sex cells) the chromosome number is n (humans: n = 23). or an individual organism.

Using cells in culture 2. Painter in 1922 was not certain whether the diploid number of humans was 46 or 48. The subsequent history of the concept can be followed in the works of Darlington and White. in contrast to their genic contents. Their behavior in animal (salamander) cells was described by Walther Flemming.3. which swells them and spreads the chromosomes . in 1882. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes.1 History of karyotype studies Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. concluding an XX/XO sex determination mechanism. Pretreating cells in a hypotonic solution. Investigation into the human karyotype took many years to settle the most basic question: how many chromosomes does a normal diploid human cell contain? In 1912. von Waldeyer in 1888. and he correctly insisted on humans having an XX/XY system. He revised his opinion later from 46 to 48. the discoverer of mitosis. these results were quite remarkable. Considering their techniques. at first favoring 46. The name was coined by another German anatomist. The next stage took place after the development of genetics in the early 20th century. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. New techniques were needed to definitively solve the problem: 1.

Arresting mitosis in metaphase by a solution of colchicines 4. The sex of an unborn fetus can be determined by observation of interphase cells. It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes. 3.2. Rather interestingly. 3. such as Giemsa.2 Observations on karyotypes 3. a suitable dye.3. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. reducing the number.2. [16] Sometimes observations may be made on non-dividing (interphase) cells. Usually. white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture.1 Staining The study of karyotypes is made possible by staining. the great apes have 48 chromosomes.2 Observations Six different characteristics of karyotypes are usually observed and compared: . is applied after cells have been arrested during cell division by a solution of colchicine. Human chromosome 2 was formed by a merger of ancestral chromosomes. For humans.

Humans have one pair fewer chromosomes than the great apes. Heterochromatin stains darker than euchromatin. type. This is brought about by translocations. permitting its loss without penalty to the organism (the dislocation hypothesis). 5. but the genes have been mostly translocated (added) to other chromosomes. This feature probably reflects different amounts of DNA duplication. 2. . faba chromosomes are many times larger. indicating tighter packing. Differences in number and position of satellites. both have six pairs of chromosomes (n=6) yet V. Differences in basic number of chromosomes may occur due to successive unequal translocations which finally remove all the essential genetic material from a chromosome. shape and banding of the chromosomes. Differences in the position of centromeres. as well as other cytogenetic information. 6. 4. 3. A full account of a karyotype may therefore include the number.1. and mainly consists of genetically inactive repetitive DNA sequences. Chromosomes can vary in absolute size by as much as twenty-fold between genera of the same family: Lotus tenuis and Vicia faba (legumes). Differences in relative size of chromosomes can only be caused by segmental interchange of unequal lengths. Differences in absolute sizes of chromosomes. which (when they occur) are small bodies attached to a chromosome by a thin thread. Differences in degree and distribution of heterochromatic regions.

Fig Diversity and evolution of karyotype Although the replication and transcription of DNA is highly standardized in eukaryotes. There is variation between species in chromosome number. Between the germ-line and soma (between gametes and the rest of the body) 3. Geographical variation between races 5.3 The human karyotype Most (but not all) species have a standard karyotype. males have both an X and a Y chromosome denoted 46. the same cannot be said for their karyotypes. 3. Between the sexes 2. Normal karyotypes for females contain two X chromosomes and are denoted 46.Variation is often found: 1. Mosaics or otherwise abnormal individuals. Between members of a population (chromosome polymorphism) 4. Any variation from the standard karyotype may lead to developmental abnormalities. XY. The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. which are highly variable. XX. and in .

despite many careful investigations. In some cases there is even significant variation within species. In some species. Chromosome elimination. This process is a carefully organised genome rearrangement where new telomeres are constructed and certain heterochromatin regions are lost.1 Changes during development Instead of the usual gene repression. In this process.. This variation provides the basis for a range of studies in evolutionary cytology. In A. karyotypic fissioning may help to explain dramatic differences in diploid numbers between closely related species. it is quite unclear what the general significance might be. Although much is known about karyotypes at the descriptive level. used in conjunction with other phylogenetic data. 3..detailed organization. despite their construction from the same macromolecules. some organisms go in for large-scale elimination of heterochromatin.3. In a review. . which were previously inexplicable. Chromatin diminution (founding father: Theodor Boveri). entire chromosomes are eliminated during development. portions of the chromosomes are cast away in particular cells. Godfrey and Masters conclude: "In our view. "We have a very poor understanding of the causes of karyotype evolution. or other kinds of visible adjustment to the karyotype. it is unlikely that one process or the other can independently account for the wide range of karyotype structures that are observed. found in some copepods and roundworms such as Ascaris suum.. But. the general significance of karyotype evolution is obscure. and it is clear that changes in karyotype organization have had effects on the evolutionary course of many species.. as in many sciarid flies.

3. all the somatic cell precursors undergo chromatin diminution. the inactivation is random as between the two Xs.2 Number of chromosomes in a set A spectacular example of variability between closely related species is the muntjac. In human females some 15% of somatic cells escape inactivation.. male = 7 chromosomes.. In marsupials it is always the paternal X which is inactivated. The diploid number of the Chinese muntjac.suum. they were astonished to find it had female = 6. which was investigated by Kurt Benirschke and his colleague Doris Wurster. The inactivation of one X chromosome takes place during the early development of mammals (see Barr body and dosage compensation). Xinactivation. The existence of supernumerary or B chromosomes .. where the haploid n = 1. the high record would be somewhere amongst the ferns. Muntiacus muntjak. In placental mammals. Muntiacus reevesi. 3. thus the mammalian female is a mosaic in respect of her X chromosomes. Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a mere 372 chromosomes. They kept quiet for two or three years because they thought something was wrong with their tissue culture. all telocentric. was found to be 46.. When they looked at the karyotype of the closely related Indian muntjac. But when they obtained a couple more specimens they confirmed [their findings]" Hsu p73-4 The number of chromosomes in the karyotype between (relatively) unrelated species is hugely variable. with the Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes. "They simply could not believe what they saw. The low record is held by the nematode Parascaris univalens.

and in some other groups. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants. occurs mainly in plants.Endopolyploidy occurs when in adult differentiated . due to the presence of five acrocentric chromosome pairs (13. Humans have FN = 82. FN. the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present. Polyploidy in animals is much less common. where one sex is diploid. and the other haploid.means that chromosome number can vary even within one interbreeding population. but it has been significant in some groups. horsetails and psilotales) is also common. and aneuploids are another example. about 70%. FN ≤ 2n. Polyploidy in lower plants (ferns.4 Ploidy Ploidy is the number of complete sets of chromosomes in a cell. 3. 21 and 22). though in this case they would not be regarded as normal members of the population. 3. It has been of major significance in plant evolution according to Stebbins. where there are more than two sets of homologous chromosomes in the cells. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%.3 Fundamental number The fundamental number.3. Polyploidy. 14. of a karyotype is the number of visible major chromosomal arms per set of chromosomes. but in grasses the average is much higher. It is a common arrangement in the Hymenoptera. Haplo-diploidy. Thus. 15. Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid.

3. Abnormalities in chromosome number usually cause a defect in development. This would give rise to a chromosome abnormality such as an extra chromosome or one or more chromosomes lost.tissues the cells have ceased to divide by mitosis. This process (especially studied in insects and some higher plants such as maize) may be a developmental strategy for increasing the productivity of tissues which are highly active in biosynthesis. it is diverse and complex. In many instances. but the nuclei contain more than the original somatic number of chromosomes. and serves differentiation and morphogenesis in many ways. The phenomenon occurs sporadically throughout the eukaryote kingdom from protozoa to man. the daughter chromosomes separating from each other inside an intact nuclear membrane.5 Aneuploidy Aneuploidy is the condition in which the chromosome number in the cells is not the typical number for the species. Down syndrome and Turner syndrome are examples of this. See palaeopolyploidy for the investigation of ancient karyotype duplications. In the endocycle (endomitosis or endoreduplication) chromosomes in a 'resting' nucleus undergo reduplication. . which is why the simple definition 'an increase in the number of chromosome sets caused by replication without cell division' is not quite accurate. endopolyploid nuclei contain tens of thousands of chromosomes (which cannot be exactly counted). The cells do not always contain exact multiples (powers of two).

3. [41] Closer to home. Human chromosome 2 was formed by a merger of ancestral chromosomes. some mantids of the genus Ameles.000 km2). Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups. reducing the number. the chromosome number is variable from one individual to another. 5. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world. that the two chromosome morphs are adapted to different habitats.500 sq mi (17. where every number from x = 3 to x = 15 is represented by at least one species. and 7. 6. 4. Well-researched examples are the ladybird beetle Chilocorus stigma.6 Species trees The detailed study of chromosome banding in insects with polytene chromosomes can reveal relationships between closely related species: the classic example is the study of chromosome banding in Hawaiian drosophilids by Hampton Carson. the great apes have 24x2 chromosomes whereas humans have 23x2. and Crocus. living from rainforests to . Classic examples in plants are the genus Crepis. 3. the European shrew Sorex araneus.Aneuploidy may also occur within a group of closely related species. There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. In about 6. where the gametic (= haploid) numbers form the series x = 3. When this happens.

it is more likely to have been a group from the same species. especially inversions. the best-studied group of Hawaiian drosophilids. Using K-Ar dating.4 million years ago (mya) (Mauna Kea) to 10mya (Necker). enabled Carson to work out the evolutionary tree long before genome analysis was practicable. All of the native Drosophila and Scaptomyza species in Hawaii have apparently descended from a single ancestral species that colonized the islands. in the family Drosophilidae. The archipelago itself (produced by the Pacific plate moving over a hot spot) has existed for far longer. In a sense. Chromosome rearrangements. when plotted in tree form (and independent of all other information). Although it would be possible for a single gravid female to colonise an island. Previous islands now beneath the sea (guyots) form the Emperor Seamount Chain. the present islands date from 0. probably 20 million years ago. at least into the Cretaceous. show a clear "flow" of species from older to newer islands. The oldest member of the Hawaiian archipelago still above the sea is Kure Atoll. The subsequent adaptive radiation was spurred by a lack of competition and a wide variety of niches. which can be dated to 30 mya. gene arrangements are visible in the banding patterns of each chromosome. make it possible to see which species are closely related. and skipping of islands. The results are clear. Drosophila and Scaptomyza. The polytene banding of the 'picture wing' group. These roughly 800 Hawaiian drosophilid species are usually assigned to two genera. The inversions. but these are much less frequent. . There are also cases of colonization back to older islands.subalpine meadows.

It yields a series of lightly and darkly stained bands .7 Depiction of karyotypes 3. late-replicating and AT rich. The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions).7. early-replicating and GC rich. • Q-banding is a fluorescent pattern obtained using quinacrine for staining. • • C-banding: Giemsa binds to constitutive heterochromatin. The pattern of bands is very similar to that seen in G-banding. adaptive radiations.the dark regions tend to be heterochromatic. so it stains centromeres.1 Types of banding Cytogenetics employs several techniques to visualize different aspects of chromosomes: • G-banding is obtained with Giemsa stain following digestion of chromosomes with trypsin. if less spectacular.There are other animals and plants on the Hawaiian archipelago which have undergone similar. . human genome. 3. R-banding is the reverse of G-banding (the R stands for "reverse"). The light regions tend to be euchromatic. • T-banding: visualize telomeres. This method will normally produce 300-400 bands in a normal.

less frequently Quinacrine. This yields a dark region where the silver is deposited. Quinacrine binds to the adeninethymine-rich regions.7. a dye. Giemsa is specific for the phosphate groups of DNA.• Silver staining: Silver nitrate stains the nucleolar organization regionassociated protein. Some karyotypes call the short and long arms p and q. often Giemsa (G-banding). denoting the activity of rRNA genes within the NOR. both chromosomes in a pair will have the same banding pattern. Karyotypes are arranged with the short arm of the chromosome on top. For example.2 Classic karyotype cytogenetics Karyogram from a human female lymphocyte probed for the Alu sequence using FISH. In addition. is used to stain bands on the chromosomes. Cri du chat syndrome involves a deletion on the short arm of . Each chromosome has a characteristic banding pattern that helps to identify them. In the "classic" (depicted) karyotype. the differently stained regions and sub-regions are given numerical designations from proximal to distal on the chromosome arms. respectively. and the long arm on the bottom. 3.

XX. It is written as 46. Short sequences of DNA from specific loci all over the genome are isolated and enumerated. allowing the visualization of the individually colored chromosomes. 3.7.chromosome 5. .8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale. a combinatorial labeling method is used to generate many different colors.3 Spectral karyotype (SKY technique) Spectral karyotyping is a molecular cytogenetic technique used to simultaneously visualize all the pairs of chromosomes in an organism in different colors.2. The critical region for this syndrome is deletion of 15. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores.5p-. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope. which is written as 46. This method is also known as virtual karyotyping. Image processing software then assigns a pseudo color to each spectrally different combination. This technique is used to identify structural chromosome aberrations in cancer cells and other disease conditions when Giemsa banding or other techniques are not accurate enough.2) 3.XX. Because there are a limited number of spectrally-distinct fluorophores.del(5)(p15.


a common chromosomal disease. are common numerical abnormalities. in which three copies of a chromosome are present instead of the usual two. Chromosomal abnormalities that lead to disease in humans include • • Turner syndrome results from a single X chromosome (45.1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical. often occur as a result of nondisjunction during meiosis in the formation of a gamete. XXY is caused by an extra X chromosome. inversions. as in derivative chromosome. X0). Also documented are trisomy 8. trisomies. is caused by trisomy of chromosome 21. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body. Numerical abnormalities. X or 45. although they generally do not survive to birth. the most common male chromosomal disease. Structural abnormalities often arise from errors in homologous recombination. • • Edwards syndrome is caused by trisomy (three copies) of chromosome 18. Klinefelter syndrome. otherwise known as 47. large-scale deletions or duplications. trisomy 9 and trisomy 16. • • Patau syndrome is caused by trisomy of chromosome 13. or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells.4. Down syndrome. translocations. including . or structural. as in the presence of extra or missing chromosomes. also known as aneuploidy. Some disorders arise from loss of just a piece of one chromosome.

a deletion of the paternal genes. There are many types of chromosome anomalies. example of imprinting disorder. a deletion of the maternal genes. abnormality or aberration reflects an atypical number of chromosomes or a structural abnormality in one or more chromosomes. numerical and structural anomalies. • • Angelman syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. from the loss of part of the short arm of chromosome 1. Chromosome anomalies usually occur when there is an error in cell division following meiosis or mitosis. A Karyotype refers to a full set of chromosomes from an individual which can be compared to a "normal" Karyotype for the species via genetic testing. a translocation mutation commonly associated with chronic myelogenous leukemia and less often with acute lymphoblastic leukemia. 1p36 Deletion syndrome. caused by abnormal formation of the larynx. . They can be organized into two basic groups. • Prader-Willi syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. from a truncated short arm on chromosome 5. Chromosomal abnormalities can also occur in cancerous cells of an otherwise genetically normal individual. example of imprinting disorder. one well-documented example is the Philadelphia chromosome. A chromosome anomaly. The name comes from the babies' distinctive cry.• Cri du chat (cry of the cat). A chromosome anomaly may be detected or confirmed in this manner.

an entire chromosome has . etc. also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21. In humans an example of a condition caused by a numerical anomaly is Down Syndrome. an X. Duplications: A portion of the chromosome is duplicated. also called the terminal 11q deletion disorder. Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome. In a Robertsonian translocation.). There are two main types of translocations. In a reciprocal translocation. Known human disorders include Charcot-Marie-Tooth disease type 1A which may be caused by duplication of the gene encoding peripheral myelin protein 22 (PMP22) on chromosome 17. segments from two different chromosomes have been exchanged. resulting in extra genetic material. Known disorders in humans include Wolf-Hirschhorn syndrome. 4.3 Structural abnormalities When the chromosome's structure is altered. which is caused by partial deletion of the short arm of chromosome 4.4. Tetrasomy. and Jacobsen syndrome. rather than two). and occurs when an individual is missing either a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (Trisomy. • • Translocations: When a portion of one chromosome is transferred to another chromosome.2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes). This can take several forms: • Deletions: A portion of the chromosome is missing or deleted.

4. Some anomalies. therefore the genetic material is inverted. • Inversions: A portion of the chromosome has broken off. 21 and 22.attached to another at the Centromere . resulting in Mosaicism (where some cells have the anomaly and some do not). and are therefore initially not inherited. the anomaly is present in every cell of the body. This is why chromosome studies are often performed on parents when a child is found to have an anomaly. 4. as well . especially the chromosomes. They often lead to an increased tendency to develop certain types of malignancies. turned upside down and reattached. Chromosome anomalies can be inherited from a parent or be "de novo".3 Inheritance Most chromosome abnormalities occur as an accident in the egg or sperm. however. • Rings: A portion of a chromosome has broken off and formed a circle or ring. 15.in humans these only occur with chromosomes 13. • Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere. It includes routine analysis of G-Banded chromosomes. Chromosome instability syndromes are a group of disorders characterized by chromosomal instability and breakage. This can happen with or without loss of genetic material. 14. Therefore.4 Cytogenetics Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell. can happen after conception. other cytogenetic banding techniques.

The name was coined by another German anatomist. Their behavior in animal (salamander) cells was described by Walther Flemming. in contrast to their genic contents. the discoverer of mitosis. 4.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). von Waldeyer in 1888. Using cells in culture 2. and he correctly insisted on man having an XX/XY system. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. Pre-treating cells in a hypotonic solution. in 1882. at first favoring 46. Painter in 1922 was not certain whether the diploid number of man was 46 or 48. which swells them and spreads the chromosomes . when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. New techniques were needed to definitively solve the problem: 1. concluding an XX/XO sex determination mechanism. Considering their techniques. He revised his opinion later from 46 to 48. The next stage took place after the development of genetics in the early 20th century. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. these results were quite remarkable. Investigation into the human karyotype took many years to settle the most basic question: how many chromosomes does a normal diploid human cell contain? In 1912.

Cutting up a photomicrograph and arranging the result into an indisputable karyogram. In 1931. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes). Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. During her cytogenetic work. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize.6 Applications in biology 4.3. 4. reducing the number. 4. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes. Arresting mitosis in metaphase by a solution of colchicine 4. Human chromosome 2 was formed by a merger of ancestral chromosomes.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. a find which eventually led to her Nobel Prize in 1983. Using Painter's technique they studied the polytene . Squashing the preparation on the slide forcing the chromosomes into a single plane 5.2 Natural populations of Drosophila In the 1930s. the great apes have 48 chromosomes. Rather interestingly.6.6. persimilis from wild populations in California and neighboring states. McClintock discovered transposons.

such as Down's syndrome. but adjust to certain frequencies at which they become stabilised.7 Human abnormalities and medical applications In the event of procedures which allowed easy enumeration of chromosomes. In 1959. It was found that the various chromosome types do not fluctuate at random. cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy. All the flies look alike whatever inversions they carry: this is an example of a cryptic polymorphism. discoveries were quickly made related to aberrant chromosomes or chromosome number. Stocks containing inversions at a known initial frequency can be maintained in controlled conditions. Evidence rapidly accumulated to show that natural selection was responsible.chromosomes and discovered that the wild populations were polymorphic for chromosomal inversions. In some congenital disorders. as with most polymorphisms. Down syndrome is also referred to as trisomy 21. This had the benefit of eliminating migration as a possible explanation of the results. Lejeune discovered patients with Down syndrome had an extra copy of chromosome 21. Using a method invented by L'Heretier and Teissier. . which enabled feeding. 4. By the time Dobzhansky published the third edition of his book in 1951 he was persuaded that the chromosome morphs were being maintained in the population by the selective advantage of the heterozygotes. Abnormalities arising from nondisjunction events can cause cells with aneuploidy (additions or deletions of entire chromosomes) in one of the parents or in the fetus. breeding and sampling whilst preventing escape. as they would if selectively neutral. Dobzhansky bred populations in population cages.

. is used today as a diagnostic for CML. Thirteen years later. has Klinefelter's Syndrome. which is required in normal females to compensate for having two copies of the chromosome. in addition to other tests. resulting in 47 total chromosomes. XYY. Identification of the Philadelphia chromosome by cytogenetics. An individual with only one sex chromosome (the X) has Turner syndrome. Pennsylvania. and XXXX. Not all genes on the X Chromosome are inactivated.as both scientists were doing their research in Philadelphia.Other numerical abnormalities discovered include sex chromosome abnormalities. the abnormal chromosome was shown by Janet Rowley to be the result of a translocation of chromosomes 9 and 22. In 1960. Many other sex chromosome combinations are compatible with live birth including XXX. The ability for mammals to tolerate aneuploidies in the sex chromosomes arises from the ability to inactivate them. with the development of more advanced techniques. Peter Nowell and David Hungerford [17] discovered a small chromosome in the white blood cells of patients with Chronic myelogenous leukemia (CML). which is why there is a phenotypic effect seen in individuals with extra X chromosomes. This abnormal chromosome was dubbed the Philadelphia chromosome . Trisomy 13 was associated with Patau's Syndrome and trisomy 18 with Edward's Syndrome. an additional X chromosome in a male.

4. Diagrams identifying the chromosomes based on the banding patterns are known as cytogenetic maps.FIG Advent of banding techniques In the late 1960s.8 Beginnings of molecular cytogenetics . Techniques were expanded to allow for culture of free amniocytes recovered from amniotic fluid. Deletions within one chromosome could also now be more specifically named and understood. Prader-Willi syndrome and others were discovered to be caused by deletions in chromosome material. Deletion syndromes such as DiGeorge syndrome. Caspersson developed banding techniques which differentially stain chromosomes. This allows chromosomes of otherwise equal size to be differentiated as well as to elucidate the breakpoints and constituent chromosomes involved in chromosome translocations. These maps became the basis for both prenatal and oncological fields to quickly move cytogenetics into the clinical lab where karyotyping allowed scientists to look for chromosomal alterations. and elongation techniques for all culture types that allow for higher resolution banding.

This change significantly increased the usage of probing techniques as fluorescent labeled probes are safer and can be used almost indefinitely.In the 1980s.1 Karyotyping . Further advances in micromanipulation and examination of chromosomes led to the technique of chromosome microdissection whereby aberrations in chromosomal structure could be isolated. advances were made in molecular cytogenetics. While radioisotope-labeled probes had been hybridized with DNA since 1969. movement was now made in using fluorescent labeled probes. Hybridizing them to chromosomal preparations using existing techniques came to be known as fluorescent in situ hybridization (FISH). cloned and studied in ever greater detail. CHAPTER 5 Techniques 5.

Routine chromosome analysis (Karyotyping) refers to analysis of metaphase chromosomes which have been banded using trypsin followed by Giemsa, Leishmanns, or a mixture of the two. This creates unique banding patterns on the chromosomes. The molecular mechanism and reason for these patterns is unknown, although it likely related to replication timing and chromatin packing. Several chromosome-banding techniques are used in cytogenetics laboratories. Quinacrine banding (Q-banding) was the first staining method used to produce specific banding patterns. This method requires a fluorescence microscope and is no longer as widely used as Giemsa banding (G-banding). Reverse banding (Rbanding) requires heat treatment and reverses the usual white and black pattern that is seen in G-bands and Q-bands. This method is particularly helpful for staining the distal ends of chromosomes. Other staining techniques include C-banding and nucleolar organizing region stains (NOR stains). These latter methods specifically stain certain portions of the chromosome. C-banding stains the constitutive heterochromatin, which usually lies near the centromere, and NOR staining highlights the satellites and stalks of acrocentric chromosomes. High-resolution banding involves the staining of chromosomes during prophase or early metaphase (prometaphase), before they reach maximal condensation. Because prophase and prometaphase chromosomes are more extended than metaphase chromosomes, the number of bands observable for all chromosomes increases from about 300 to 450 to as many as 800. This allows the detection of less obvious abnormalities usually not seen with conventional banding.

5.2 Slide preparation Cells from bone marrow, blood, amniotic fluid, cord blood, tumor, and tissues (including skin, umbilical cord, chorionic villi, liver, and many other organs) can be cultured using standard cell culture techniques in order to increase their number. A mitotic inhibitor (colchicine, colcemid) is then added to the culture. This stops cell division at mitosis which allows an increased yield of mitotic cells for analysis. The cells are then centrifuged and media and mitotic inhibitor are removed, and replaced with a hypotonic solution. This causes the white blood cells or fibroblasts to swell so that the chromosomes will spread when added to a slide as well as lyses the red blood cells. After the cells have been allowed to sit in hypotonic, Carnoy's fixative (3:1 methanol to glacial acetic acid) is added. This kills the cells and hardens the nuclei of the remaining white blood cells. The cells are generally fixed repeatedly to remove any debris or remaining red blood cells. The cell suspension is then dropped onto specimen slides. After aging the slides in an oven or waiting a few days they are ready for banding and analysis. 5.3 Analysis Analysis of banded chromosomes is done at a microscope by a clinical laboratory specialist in cytogenetics (CLSp(CG)). Generally 20 cells are analyzed which is enough to rule out mosaicism to an acceptable level. The results are summarized and given to a board-certified cytogeneticist for review, and to write an interpretation taking into account the patients previous history and other clinical findings. The results are then given out reported in an International System for Human Cytogenetic Nomenclature 2009 (ISCN2009). 5.4 Fluorescent in situ hybridization

Fluorescent in situ hybridization refers to using fluorescently labeled probe to hybridize to cytogenetic cell preparations. In addition to standard preparations FISH can also be performed on:
• • • • • • •

bone marrow smears blood smears paraffin embedded tissue preparations enzymatically dissociated tissue samples uncultured bone marrow uncultured amniocytes cytospin preparations

5.5 Slide preparation The slide is aged using a salt solution usually consisting of 2X SSC (salt, sodium citrate). The slides are then dehydrated in ethanol, and the probe mixture is added. The sample DNA and the probe DNA are then co-denatured using a heated plate and allowed to re-anneal for at least 4 hours. The slides are then washed to remove excess unbound probe, and counterstained with 4',6-Diamidino-2phenylindole (DAPI) or propidium iodide. 5.7 Analysis Analysis of FISH specimens is done by fluorescence microscopy by a clinical laboratory specialist in cytogenetics. For oncology generally a large number of interphase cells are scored in order to rule out low level residual disease,

you can solve technical computing problems faster than with traditional programming languages. and computational biology. . For congenital problems usually 20 metaphase cells are scored. and numerical computation. test and measurement. CHAPTER 6 MATLAB MATLAB is a high-level technical computing language and interactive environment for algorithm development. Add-on toolboxes (collections of special-purpose MATLAB functions) extend the MATLAB environment to solve particular classes of problems in these application areas. such as comparative genomic hybridization arrays. data analysis. such as C.generally between 200 and 1000 cells are counted and scored. Future of cytogenetics Advances now focus on molecular cytogenetics including automated systems for counting the results of standard FISH preparations and techniques for virtual karyotyping. C++. communications. data visualization. financial modeling and analysis. You can use MATLAB in a wide range of applications. CGH and Single nucleotide polymorphism-arrays. including signal and image processing. and FORTRAN. Using MATLAB. control design.

The image brightness and contrast depend on the specific tuning of the microscope and the particular geometric shape of each chromosome depends on the specific metaphase plaque from which the chromosomes were extracted. MATLAB eliminates the need for ‘for’ loops. It enables fast development and execution. and allocating memory. 6.1 Image Processing The image processing step aims at image contrast enhancement and compensation of geometric distortions observed in each chromosome not related with its intrinsic shape or size. such as declaring variables. The image processing step is composed of the following operations. and distribute your MATLAB algorithms and applications. In many cases. These effects must be compensated to improve the results of the pairing algorithm. The MATLAB language supports the vector and matrix operations that are fundamental to engineering and scientific problems. one line of MATLAB code can often replace several lines of C or C++ code. With the MATLAB language. As a result. You can integrate your MATLAB code with other languages and applications. specifying data types. you can program and develop algorithms faster than with traditional languages because you do not need to perform lowlevel administrative tasks. .MATLAB provides a number of features for documenting and sharing your work.

Therefore. or at least attenuated. 2) Geometrical compensation—The geometric compensation. performed by using the algorithm is needed to obtain chromosomes with vertical medial axis. To compare chromosomes from a band pattern point of view.2 Concepts used in this phase 1) Image conversion 2) Denoising . the spatially scaled images are histogram equalized. 6.1) Chromosome extraction—Each chromosome is isolated from the unordered karyogram. geometrical and dimensional differences must be removed. To compensate for this inhomogeneity. a dimensional scaling is performed before the pattern features is extracted to make all the chromosome with the same size and aspect ratio by interpolating the original images. 4) Intensity compensation—The metaphase plaque from which the chromosomes are extracted does not present a uniform brightness and contrast. This compensation algorithm is composed of the following main steps: a) chromosome and medial axis segmentation b) axis smoothing c) interpolationalong orthogonal lines to the smoothed medial axis d) border regularization 3) Shape normalization—The features used in the comparison of chromosomes are grouped into two classes: 1) geometric based 2) pattern based (G-banding).

I). if you want to filter a color image that is stored as an indexed image. If you attempt to filter the indexed image. MATLAB filters the intensity values in the image. so the image displays as shades of gray.I. green. . RGB = cat (3. the region of interest functions returns a binary image that you can use to mask an image for filtering or for other operations. The resulting true color image has identical matrices for the red. For example. For example. you must first convert it to true color format. In addition to these image type conversion functions.I.2. When you apply the filter to the true color image. listed in the following table. you can convert a grayscale image to true color format by concatenating three copies of the original matrix along the third dimension. For example. and blue planes. 6. and the results might not be meaningful. MATLAB simply applies the filter to the indices in the indexed image matrix. there are other functions that return a different image type as part of the operation they perform. as is appropriate.3) Edge detection 4) Two dimensional convolutions.1 Image conversion The toolbox includes many functions that you can use to convert an image from one type to another. You can perform certain conversions just using MATLAB syntax.

We may use edges to measure the size of objects in an image. These errors will appear on the image output in different ways depending on the type of disturbance in the signal. . to recognize or classify objects. Usually we know what type of errors to expect. we may expect errors to occur in the image signal. to isolate particular objects from their background.'method'. If an image is being sent electronically from one place to another.3 Two dimensional convolutions C = conv2(A. or through networked cable.2. Cleaning an image corrupted by noise is thus an important area of image restoration.2. ) Where the parameters available depend on the method used 6. hence we can choose the most appropriate method for reducing the effects.6. . If one of these matrices describes a two-dimensional finite impulse response .parameters. and hence the type of noise on the image. The general Matlab command for finding edges is edge(image. and we shall look at some of the more straightforward of them. 6.B) computes the two-dimensional convolution of matrices A and B. There is a large number of edge finding algorithms in existence.5 Edge detection Edges contain some of the most useful information in an image. via satellite or wireless transmission. caused by external disturbance.4 Denoising We may define noise to be any degradation in the image signal.

na] and the size of B is [mb.A) C in each dimension is equal to the sum of the corresponding dimensions of the input A is [ma.hrow. C = conv2(. .'shape') subsection of the two-dimensional convolution. convolves A first with the vector hcol along the rows and then returns a with the vector hrow along the columns. as specified by the shape parameter Algorithms description 1) Read the image and convert into gray 2)Remove noise 3) Background separation 4) Edge detect 5) Separate the pairs MODULE 1 PSEUDO CODE iimread('12345. the other matrix is filtered in two dimensions. if the size of then the size of C is [ma+mb-1.na+nb-1].(FIR) filter. imedfilt2(im1.. That is. If hcol is a column vector and hrow is a row vector...bmp'). The size of matrices.[3 3]). rgb2gray im2bw(im.A).'sobel'). this case is the same as C = conv2(hcol*hrow.nb]. minus one.0. edge(im1. The indices of the center element of B are defined as floor(([mb C = conv2(hcol.7). nb]+1)/2).

bwlabel(B.[imx.imy]=size(BW).8). [r. y1=rc(i. . Msk conv2(double(BW).j)==L_number(k) flag=1. flag=0.c] = find(L==22). L_number=zeros(mx.n]=size(L).1). nzeros(imx. for i=1:m for j=1:n if L(i. rc = [r c].double(msk)). n1(x1.2). MODULE 2 clc [m.j)~=0 for k=1:mx if L(i. Index=1.1). for i=1:sx x1=rc(i. mx=max(max(L)). [sx sy]=size(rc).y1)=255.imy).

.41.40.57.y1)=255.20. [sx sy]=size(rc).56.39.1).50. Test_number=[3. 36.51.32.[]).52.8.end end if flag~=1 L_number(Index)=L(i.7. y1=rc(i. rc = [r c].33.48.29. end %h=figure.6. for i=1:sx x1=rc(i. n1=zeros(imx.j). Index=Index+1.19. n1(x1.10.54.imshow(n1. end.66].14.2).c] = find(L==L_number((Test_number(x)))).49. end flag=0.28.22. end end L_number.35. for x=1:46 [r.31.

s1=bwmorph(s. BW=im2bw(f).1. skel=im2bw(skel. Circumference_sum=0.'. Area=zeros(46.Inf). Arm_length_sum=0. .'skel'. BW=double(BW). end end end Circumference(i)=Circumference_sum.'spur'. s=bwmorph(skel.y)==1 Circumference_sum=Circumference_sum+1. Arm_length=zeros(46. skel=im2double(f). for i=1:46 f=imread(strcat(num2str(i). BW1=edge(BW. f=imcomplement(f).8).1).5*graythresh(skel)).'canny').end Circumference=zeros(46. for x=1:m for y=1:n if BW1(x. [m n]=size(BW1).1).1).bmp')).

Arm_length. for x=1:m for y=1:n if s1(x. for x=1:m for y=1:n if BW(x. BW=im2bw(f). end end end Area(i)=Area_sum.y)==1 Arm_length_sum=Arm_length_sum+1. end Circumference. .[m n]=size(s1). [m n]=size(BW). end end end Arm_length(i)=Arm_length_sum. Area_sum=0.y)==1 Area_sum=Area_sum+1.

1)=46.Area. for j=1:46 if i~=j && abs(Circumference(i)-Circumference(j))+abs(Arm_length(i)Arm_length(j))+abs(Area(i)-Area(j))<min min=abs(Circumference(i)-Circumference(j))+abs(Arm_length(i)Arm_length(j))+abs(Area(i)-Area(j)). end end Pair. Pair=zeros(46. Pair(46.2)=j.2)==46 Pair(46.2). Pair(i. end end end for i=1:45 if Pair(i.2)=i.1)=i. Pair(i.2)=i+1. . Pair(i.1)=i. Pair(i. for i=1:45 min=abs(Circumference(i)-Circumference(i+1))+abs(Arm_length(i)Arm_length(i+1))+abs(Area(i)-Area(i+1)).

end f1=imread(strcat(num2str(Pair(i. .2.'. end flag=0.delete=zeros(46.1)). imshow(f1). end f2=imread(strcat(num2str(Pair(i.figure_flag). imshow(f2). if figure_flag~=47 subplot(23.'. figure_flag=figure_flag+1.2.bmp')).2)).1)==delete(j) flag=1.2). flag=0. delete(figure_flag)=Pair(i.bmp')). figure_flag=figure_flag+1.figure_flag). end end if flag~=1 if figure_flag~=47 subplot(23. figure_flag=1. for i=1:46 for j=1:46 if Pair(i.1).

end CONCLUTION In this paper. such as. and Philadelphia. The images of these chromosomes present less quality and level of detail than the ones usually used in traditional genetic analysis using datasets such as Edinburgh. a newmetric is proposed to measure the distance between chromosomes to be used in the automatic chromosome pairing procedure. The algorithm is composed by four main steps: 1) image processing of the karyograms provided by the technicians. dimensions and banding profiles. plus a new one. in the scope of karyotyping process used in cytogentic analysis. The main goal of this paper is to provide useful contributions toward the design of a fully automatic chromosomes pairing algorithm of bone marrow cells to be used in the diagnosis of leukemia. Copenhagen. 2) feature extraction from the processed images . based on the MI. to improve the discriminative power of the pairing algorithm with respect to the the G-banding pattern. The proposed algorithm is based on the traditional features extracted from the karyogram.

Vectors of coefficients associated with each one of the 22 classes are computed and the distance between two arbitrary chromosomes is the minimum one among all distances obtained with these 22 vectors. a novel metric distance is proposed to be used in the pairing procedure that linearly combines the distances associated with each feature. by minimizing the overall distances between chromosomes of the same class (intraclass) and by maximizing the distances between chromosomes when at least one of them does not belong to that class (interclass). from the chromosomes in the training set. The features extracted from the processed images discriminate each pair with respect to their size. This normalization is needed to make it possible the band pattern comparison between chromosomes. shape. the romosome images. Tests using 19 karyograms based on bone marrow cells. achieves a 70. and band pattern. The coefficients of the linear combination are obtained through a training step using chromosomes paired manually by experts. 3) training of a classifier (performed once) where similarity among chromosomes are characterized. In the image processing step. extracted from the unordered karyogram. Here. The training process consists in the estimation of each vector of coefficient . and finally. The addition of the MI feature to the traditional geometrical and band profile features described in the literature leads to a clear improvement in the .10% mean classification rate. shape and band pattern. 4) pairing.characterizing the size. and to normalize their dimensions. are processed in order to compensate for geometrical and intensity distortions.working with 22 classes of chromosomes and a LOOCV strategy allowus to conclude that the proposed pairing algorithms. The pairing process is performed by efficiently solving a combinatorial problem where a permutation matrix is obtained from the distance matrix computed with the extracted features associated with each pair of chromosomes in the karyogram.working within an 8-D feature space.

g. REFERENCES . a new chromosome dataset with 9200 chromosomes from bone marrow cells. or Philadelphia. despite the low quality of this type of chromosomes. Copenhagen. The results presented in this paper are promising. Using 27 karyograms andworking with a limited number of classes (≤ 8). it was shown that it is possible to achieve comparable classification rates to the ones obtained with the classical chromosome dataset. In addition. This dataset was made publicly available [29]. and from which it is possible to extract additional features. Qualitative comparisons with the results obtained with the Leica CW 4000 Karyopairing software using the same data were performed and have shown relevant improvements. amean classification rate larger than 93% was obtained in all experiments. called LK1 . Executing the algorithm on a higher quality dataset. such as Edinburgh..10% classification ratewas obtained. e. presenting a uniform level of condensation. was built to provide a ground truth to test classification and pairing algorithms for this type of “low” image quality chromosomes. whose images are of significantly higher quality. centromere position. In fact.performance of the classifier. a 76.

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