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

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

3 MUTATIONS IN CHROMOSOME NUMBER Normally. rather than 2. An example of aneuploidy is trisomy 21. a extra copy of human chromosome 21).XY or 47. 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). If the mutation involves only one or a few chromosomes in the genome (e. 1. Such individuals are called euploid and have the wild-type chromosome complement for the species. Euploid human karyotypes are 46. The individual would have Down Syndrome and his/her karyotype would be written 47.g. in which an individual has 3. . the individual carrying the mutation is said to be aneuploid.+21.XX. reflecting their bacterial origins.+21. copies of chromosome 21.are also found in mitochondria and chloroplasts. 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. The simplest gonophores are found in viruses: these DNA or RNA molecules are short linear or circular gonophores that often lack structural proteins. XX (female) or 46 XY (male).

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

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

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

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

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

However. continues to grow as it duplicates its chromosomes (S).1 Preprophase In plant cells only. During all three phases. S (synthesis). chromosomes are replicated only during the S phase. where the cell prepares itself for cell division. This is achieved through the formation of a phragmosome. It alternates with the much longer interphase. grows more and prepares for mitosis (G 2). All these phases in the interphase are highly regulated.2. and finally it divides (M) before restarting the cycle. mainly via proteins. the cell grows by producing proteins and cytoplasmic organelles. a transverse sheet of cytoplasm that bisects the cell along the future plane of cell . prophase is preceded by a pre-prophase stage. 2. In highly vacuolated plant cells. 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. the nucleus has to migrate into the center of the cell before mitosis can begin. Thus. Interphase is divided into three phases: G1 (first gap). and G2 (second gap).2.2 Phases of cell cycle and mitosis Interphase Fig 4 The cell cycle The mitotic phase is a relatively short period of the cell cycle. a cell grows (G1).

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

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

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

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

allowing them to separate. the nonkinetochore microtubules elongate.4 Anaphase When every kinetochore is attached to a cluster of microtubules and the chromosomes have lined up along the metaphase plate. Two events then occur: first.” “against. These sister chromatids.” “back. At the end of anaphase. although there is a theory that suggests that the rapid assembly and breakdown of microtubules may cause this movement.Because proper chromosome separation requires that every kinetochore be attached to a bundle of microtubules (spindle fibres). The force that causes the centrosomes to move towards the ends of the cell is still unknown. The signal creates the mitotic spindle checkpoint. which have now become distinct sister chromosomes. 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. 2. Early anaphase is usually defined as the separation of the sister chromatids. Next. the proteins that bind sister chromatids together are cleaved. the cell proceeds to anaphase (from the Greek meaning “up. the cell has succeeded in separating identical copies of the genetic material into two distinct populations. These two stages are sometimes called early and late anaphase. pulling the centrosomes (and the set of chromosomes to which they are attached) apart to opposite ends of the cell. .” or “re-”). are pulled apart by shortening kinetochore microtubules and move toward the respective centrosomes to which they are attached.

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

2.3 Cell replacement In some parts of body. The phragmoplast is a microtubule structure typical for higher plants. 2. This is the basis of the development of a multicellular body from a single cell i. The end of cytokinesis marks the end of the M-phase.e.2 Development and growth The number of cells within an organism increases by mitosis.1Significance Mitosis is important for the maintenance of the chromosomal set. Following are the occasions in the lives of organism where mitosis happens: 2. whereas some green algae use a phycoplast microtubule array during cytokinesis. zygote and also the basis of the growth of a multicellular body.division is also driven by vesicles derived from the Golgi apparatus. separating the two nuclei. New cells are formed by mitosis and so are exact copies of the cells being replaced.4 Regeneration .5.5. skin and digestive tract. Each daughter cell has a complete copy of the genome of its parent cell. Similarly. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall.5.g. e. which move along microtubules to the middle of the cell. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis.. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell. 2. cells are constantly sloughed off and replaced by new ones.5.

especially during early cellular divisions in the zygote. Mitosis continues in the cells of bud and it grows into a new individual. a condition known as trisomy. For example. For example.5. the hydra reproduces asexually by budding. The cells at the surface of hydra undergo mitosis and form a mass called bud. they fail to complete cell division and retain both nuclei in one cell. a condition known as monosomy.7 Consequences of errors Although errors in mitosis are rare. Occasionally when cells experience nondisjunction. The same division happens during asexual reproduction or vegetative propagation in plants. sea star regenerates its lost arm through mitosis. The production of new cells is achieved by mitosis. a chromosome may fail to separate during anaphase. Mitotic errors can be especially dangerous to the organism because future offspring from this parent cell will carry the same disorder. the process may go wrong. These cells are considered aneuploid. and the latter cell having only one chromosome (the homologous chromosome). One daughter cell will receive both sister chromosomes and the other will receive none. resulting in binucleated cells.6 Asexual reproduction Some organisms produce genetically similar offspring through asexual reproduction. a condition often associated with cancer. 2.5. 2. In non-disjunction. This results in the former cell having three chromosomes containing the same genes (two sisters and a homologue).Some organisms can regenerate their parts of bodies. .

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

Early events of metaphase can . 2. microtubules formed in prophase have already found and attached themselves to kinetochores in metaphase. The centromeres of the chromosomes convene themselves on the metaphase plate (or equatorial plate). Preceded by events in prometaphase and followed by anaphase.7 Metaphase Metaphase. is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes. An example of a cell that goes through endomitosis is the megakaryocyte. In certain types of cells. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. carrying genetic information. only roughly lining up along the middleline. resulting in cells with many copies of the same chromosome occupying a single nucleus. Metaphase accounts for approximately 4% of the cell cycle's duration. an imaginary line that is equidistant from the two centrosome poles.6 Endomitosis Endomitosis is a variant of mitosis without nuclear or cellular division. This process may also be referred to as endoreduplication and the cells as endoploid. from the ancient Greek(between) and (stage). chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. analogous to a tug of war between equally strong people. align in the middle of the cell before being separated into each of the two daughter cells.2.

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

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

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

3. He revised his opinion later from 46 to 48. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. at first favoring 46. in 1882. 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. and he correctly insisted on humans having an XX/XY system. New techniques were needed to definitively solve the problem: 1. Pretreating cells in a hypotonic solution. The name was coined by another German anatomist. these results were quite remarkable. Considering their techniques. 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. in contrast to their genic contents. Their behavior in animal (salamander) cells was described by Walther Flemming. which swells them and spreads the chromosomes . von Waldeyer in 1888. the discoverer of mitosis. The next stage took place after the development of genetics in the early 20th century. Using cells in culture 2. concluding an XX/XO sex determination mechanism.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.

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

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

Geographical variation between races 5. Between members of a population (chromosome polymorphism) 4. which are highly variable.3 The human karyotype Most (but not all) species have a standard karyotype. Any variation from the standard karyotype may lead to developmental abnormalities.Variation is often found: 1. and in . Between the sexes 2. Between the germ-line and soma (between gametes and the rest of the body) 3. 3. XY. Normal karyotypes for females contain two X chromosomes and are denoted 46. the same cannot be said for their karyotypes. The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. Mosaics or otherwise abnormal individuals. males have both an X and a Y chromosome denoted 46. 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. XX.

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

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

Endopolyploidy occurs when in adult differentiated . Polyploidy. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants. of a karyotype is the number of visible major chromosomal arms per set of chromosomes. Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid. due to the presence of five acrocentric chromosome pairs (13.3 Fundamental number The fundamental number. and aneuploids are another example. though in this case they would not be regarded as normal members of the population. the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present. FN ≤ 2n. where there are more than two sets of homologous chromosomes in the cells. It has been of major significance in plant evolution according to Stebbins. Polyploidy in lower plants (ferns. but in grasses the average is much higher. 3. 14. Haplo-diploidy.4 Ploidy Ploidy is the number of complete sets of chromosomes in a cell. where one sex is diploid. and the other haploid.means that chromosome number can vary even within one interbreeding population. Thus. Polyploidy in animals is much less common. Humans have FN = 82. but it has been significant in some groups. horsetails and psilotales) is also common. 3. and in some other groups. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%. 15. FN. It is a common arrangement in the Hymenoptera. occurs mainly in plants. 21 and 22).3. about 70%.

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

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. 4. There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast. and 7.500 sq mi (17.Aneuploidy may also occur within a group of closely related species. the chromosome number is variable from one individual to another. where every number from x = 3 to x = 15 is represented by at least one species. [41] Closer to home. 3.000 km2). Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world. where the gametic (= haploid) numbers form the series x = 3. Human chromosome 2 was formed by a merger of ancestral chromosomes. living from rainforests to . 6. the great apes have 24x2 chromosomes whereas humans have 23x2. Well-researched examples are the ladybird beetle Chilocorus stigma. reducing the number. some mantids of the genus Ameles.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. and Crocus. the European shrew Sorex araneus. that the two chromosome morphs are adapted to different habitats. 5. When this happens. 3. Classic examples in plants are the genus Crepis. In about 6.

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

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

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

Short sequences of DNA from specific loci all over the genome are isolated and enumerated.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale. It is written as 46. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores. This method is also known as virtual karyotyping. Image processing software then assigns a pseudo color to each spectrally different combination.del(5)(p15.XX. a combinatorial labeling method is used to generate many different colors.chromosome 5.2) 3.7.5p-. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope.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. The critical region for this syndrome is deletion of 15. Because there are a limited number of spectrally-distinct fluorophores.2.XX. 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. which is written as 46. . 3. allowing the visualization of the individually colored chromosomes.


often occur as a result of nondisjunction during meiosis in the formation of a gamete. otherwise known as 47. • • Patau syndrome is caused by trisomy of chromosome 13. trisomy 9 and trisomy 16. Klinefelter syndrome. trisomies. in which three copies of a chromosome are present instead of the usual two. or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells. are common numerical abnormalities.1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical. including . a common chromosomal disease. is caused by trisomy of chromosome 21. Structural abnormalities often arise from errors in homologous recombination. X or 45. although they generally do not survive to birth. large-scale deletions or duplications. as in derivative chromosome. Down syndrome. Some disorders arise from loss of just a piece of one chromosome. the most common male chromosomal disease. as in the presence of extra or missing chromosomes.4. X0). inversions. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body. Chromosomal abnormalities that lead to disease in humans include • • Turner syndrome results from a single X chromosome (45. Numerical abnormalities. translocations. Also documented are trisomy 8. XXY is caused by an extra X chromosome. or structural. • • Edwards syndrome is caused by trisomy (three copies) of chromosome 18. also known as aneuploidy.

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

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. and Jacobsen syndrome. rather than two). an X. segments from two different chromosomes have been exchanged. There are two main types of translocations.2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes). also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21. In a reciprocal translocation. This can take several forms: • Deletions: A portion of the chromosome is missing or deleted. 4. Tetrasomy. which is caused by partial deletion of the short arm of chromosome 4.).4. an entire chromosome has . Known disorders in humans include Wolf-Hirschhorn syndrome. also called the terminal 11q deletion disorder. In a Robertsonian translocation. Duplications: A portion of the chromosome is duplicated. • • Translocations: When a portion of one chromosome is transferred to another chromosome. resulting in extra genetic material. Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome. and occurs when an individual is missing either a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (Trisomy. In humans an example of a condition caused by a numerical anomaly is Down Syndrome. etc.3 Structural abnormalities When the chromosome's structure is altered.

This is why chromosome studies are often performed on parents when a child is found to have an anomaly. 21 and 22.in humans these only occur with chromosomes 13.4 Cytogenetics Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell. other cytogenetic banding techniques. especially the chromosomes. 4. 14.3 Inheritance Most chromosome abnormalities occur as an accident in the egg or sperm. Some anomalies. Chromosome instability syndromes are a group of disorders characterized by chromosomal instability and breakage. This can happen with or without loss of genetic material. as well . turned upside down and reattached. can happen after conception. and are therefore initially not inherited. 4. therefore the genetic material is inverted. however. Chromosome anomalies can be inherited from a parent or be "de novo". • Rings: A portion of a chromosome has broken off and formed a circle or ring. Therefore. • Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere.attached to another at the Centromere . They often lead to an increased tendency to develop certain types of malignancies. • Inversions: A portion of the chromosome has broken off. It includes routine analysis of G-Banded chromosomes. the anomaly is present in every cell of the body. 15. resulting in Mosaicism (where some cells have the anomaly and some do not).

when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. 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. these results were quite remarkable. the discoverer of mitosis. Painter in 1922 was not certain whether the diploid number of man was 46 or 48. Considering their techniques. Pre-treating cells in a hypotonic solution. in contrast to their genic contents.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). He revised his opinion later from 46 to 48. The name was coined by another German anatomist. in 1882. Their behavior in animal (salamander) cells was described by Walther Flemming.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. Using cells in culture 2. which swells them and spreads the chromosomes . 4. 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. at first favoring 46. New techniques were needed to definitively solve the problem: 1. von Waldeyer in 1888. 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. concluding an XX/XO sex determination mechanism.

Squashing the preparation on the slide forcing the chromosomes into a single plane 5. a find which eventually led to her Nobel Prize in 1983. 4. During her cytogenetic work.3. 4.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. Rather interestingly.6. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes).2 Natural populations of Drosophila In the 1930s. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize. McClintock discovered transposons. Arresting mitosis in metaphase by a solution of colchicine 4. Using Painter's technique they studied the polytene . the great apes have 48 chromosomes. In 1931.6.6 Applications in biology 4. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. Human chromosome 2 was formed by a merger of ancestral chromosomes. persimilis from wild populations in California and neighboring states. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes. reducing the number. Cutting up a photomicrograph and arranging the result into an indisputable karyogram.

Dobzhansky bred populations in population cages. as they would if selectively neutral.chromosomes and discovered that the wild populations were polymorphic for chromosomal inversions. 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. In some congenital disorders. Using a method invented by L'Heretier and Teissier. Down syndrome is also referred to as trisomy 21. discoveries were quickly made related to aberrant chromosomes or chromosome number. which enabled feeding. Stocks containing inversions at a known initial frequency can be maintained in controlled conditions. 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. All the flies look alike whatever inversions they carry: this is an example of a cryptic polymorphism.7 Human abnormalities and medical applications In the event of procedures which allowed easy enumeration of chromosomes. cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy. as with most polymorphisms. such as Down's syndrome. Evidence rapidly accumulated to show that natural selection was responsible. It was found that the various chromosome types do not fluctuate at random. 4. In 1959. . 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. breeding and sampling whilst preventing escape. but adjust to certain frequencies at which they become stabilised.

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

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

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

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,

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

These effects must be compensated to improve the results of the pairing algorithm. one line of MATLAB code can often replace several lines of C or C++ code. MATLAB eliminates the need for ‘for’ loops. In many cases.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. 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. 6. you can program and develop algorithms faster than with traditional languages because you do not need to perform lowlevel administrative tasks. specifying data types.MATLAB provides a number of features for documenting and sharing your work. . such as declaring variables. The image processing step is composed of the following operations. The MATLAB language supports the vector and matrix operations that are fundamental to engineering and scientific problems. You can integrate your MATLAB code with other languages and applications. As a result. With the MATLAB language. and allocating memory. It enables fast development and execution. and distribute your MATLAB algorithms and applications.

6. 4) Intensity compensation—The metaphase plaque from which the chromosomes are extracted does not present a uniform brightness and contrast.2 Concepts used in this phase 1) Image conversion 2) Denoising .1) Chromosome extraction—Each chromosome is isolated from the unordered karyogram. Therefore. To compensate for this inhomogeneity. geometrical and dimensional differences must be removed. the spatially scaled images are histogram equalized. To compare chromosomes from a band pattern point of view. 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). 2) Geometrical compensation—The geometric compensation. performed by using the algorithm is needed to obtain chromosomes with vertical medial axis. 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. or at least attenuated.

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

6.parameters. Cleaning an image corrupted by noise is thus an important area of image restoration. .'method'. we may expect errors to occur in the image signal. The general Matlab command for finding edges is edge(image. to recognize or classify objects.2.2. There is a large number of edge finding algorithms in existence. to isolate particular objects from their background. We may use edges to measure the size of objects in an image. 6. and we shall look at some of the more straightforward of them. and hence the type of noise on the image. . These errors will appear on the image output in different ways depending on the type of disturbance in the signal. hence we can choose the most appropriate method for reducing the effects. ) Where the parameters available depend on the method used 6. or through networked cable. via satellite or wireless transmission. Usually we know what type of errors to expect. If an image is being sent electronically from one place to another.5 Edge detection Edges contain some of the most useful information in an image.B) computes the two-dimensional convolution of matrices A and B. If one of these matrices describes a two-dimensional finite impulse response . caused by external disturbance.4 Denoising We may define noise to be any degradation in the image signal.3 Two dimensional convolutions C = conv2(A.

0.A).A) C in each dimension is equal to the sum of the corresponding dimensions of the input A is [ma. nb]+1)/2).hrow. 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. If hcol is a column vector and hrow is a row vector.[3 3]).bmp').na] and the size of B is [mb. minus one.. the other matrix is filtered in two dimensions.na+nb-1]. convolves A first with the vector hcol along the rows and then returns a with the vector hrow along the columns. That is... C = conv2(.(FIR) filter.7). The size of matrices.nb].'shape') subsection of the two-dimensional convolution. this case is the same as C = conv2(hcol*hrow. imedfilt2(im1. edge(im1. if the size of then the size of C is [ma+mb-1. . The indices of the center element of B are defined as floor(([mb C = conv2(hcol. rgb2gray im2bw(im.'sobel').

c] = find(L==22). for i=1:m for j=1:n if L(i. [sx sy]=size(rc). mx=max(max(L)).2). Index=1. . flag=0. bwlabel(B.j)~=0 for k=1:mx if L(i.imy). [r.[imx.imy]=size(BW). nzeros(imx. y1=rc(i.1). L_number=zeros(mx.y1)=255.1). Msk conv2(double(BW).double(msk)).n]=size(L).8). n1(x1. for i=1:sx x1=rc(i.j)==L_number(k) flag=1. MODULE 2 clc [m. rc = [r c].

48.10.imshow(n1.20.31. for i=1:sx x1=rc(i.51.56.15. . [sx sy]=size(rc).32. end flag=0.21. end %h=figure. n1(x1. Index=Index+1.40.j). rc = [r c]. n1=zeros(imx.y1)=255.end end if flag~=1 L_number(Index)=L(i. for x=1:46 [r.]. y1=rc(i.57.65.[]).43.14.c] = find(L==L_number((Test_number(x)))). Test_number=[ end. end end L_number.

1).8). BW=double(BW). [m n]=size(BW1).y)==1 Circumference_sum=Circumference_sum+1. s=bwmorph(skel. f=imcomplement(f). BW1=edge(BW.'skel'.'canny'). BW=im2bw(f). Arm_length_sum=0.bmp')). skel=im2bw(skel. .end Circumference=zeros(46.Inf).5*graythresh(skel)). skel=im2double(f). end end end Circumference(i)=Circumference_sum. s1=bwmorph(s.1. for x=1:m for y=1:n if BW1(x. for i=1:46 f=imread(strcat(num2str(i).'spur'. Arm_length=zeros(46.1).1). Area=zeros(46. Circumference_sum=0.'.

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

Pair(46. end end Pair. 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)).2)=i+1.2)=i. 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)).Area. end end end for i=1:45 if Pair(i.2)==46 Pair(46.2).2)=j. Pair(i.1)=i.1)=i. Pair=zeros(46. Pair(i. Pair(i. . Pair(i.1)=46.

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

The proposed algorithm is based on the traditional features extracted from the karyogram.end CONCLUTION In this paper. dimensions and banding profiles. in the scope of karyotyping process used in cytogentic analysis. based on the MI. to improve the discriminative power of the pairing algorithm with respect to the the G-banding pattern. 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. plus a new one. Copenhagen. 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. such as. and Philadelphia. a newmetric is proposed to measure the distance between chromosomes to be used in the automatic chromosome pairing procedure. 2) feature extraction from the processed images . The algorithm is composed by four main steps: 1) image processing of the karyograms provided by the technicians.

and to normalize their dimensions. the romosome images. are processed in order to compensate for geometrical and intensity distortions. extracted from the unordered karyogram. The training process consists in the estimation of each vector of coefficient .working with 22 classes of chromosomes and a LOOCV strategy allowus to conclude that the proposed pairing algorithms. 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. Here. 3) training of a classifier (performed once) where similarity among chromosomes are characterized. and band pattern.10% mean classification rate. from the chromosomes in the training set. 4) pairing. shape and band pattern. 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).characterizing the size. a novel metric distance is proposed to be used in the pairing procedure that linearly combines the distances associated with each feature. shape. achieves a 70. 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 . In the image processing step. 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. This normalization is needed to make it possible the band pattern comparison between chromosomes. The features extracted from the processed images discriminate each pair with respect to their size. The coefficients of the linear combination are obtained through a training step using chromosomes paired manually by experts. Tests using 19 karyograms based on bone marrow cells.

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

Chapter XII: The Karyotype. 19-174. preconception and the counting of the human chromosomes. 1931. Res. 147–49. 1973. ^ Concise Oxford Dictionary London. ^ Levitsky G. Arch. 1922. The chromosomes. Med. From 48 to 46: cytological technique. 1950. Variation and evolution in plants.L. Anat. biologie 27.. ^ a b c White M. Evolution of genetic systems. 9. Cambridge University Press. Etudes sur la spermatogenese humaine. revised and enlarged.D. The material basis of heredity. Bull. 1912. Stansfield W. 2nd ed. Animal cytology and evolution. 7th ed. Hist. p. 6th ed. Oxford U. 10. Oliver & Boyd.C.S. ^ Darlington C. ^ von Winiwarter H.J. p242 5. ^ Kottler M. 129. ^ Levitsky G. 48. . 1939. Bull. 465-502. ^ King R. ^ a b White M. and Mulligan P. Cambridge University Press. The morphology of chromosomes.1. 28 2. State Publication Office of the Ukraine. [in Russian] 6. 1924. 1974. Genet.A. Applied Bot. 23. 8. 7.D. A dictionary of genetics.K.D. Chapman & Hall. 3rd ed.D. The spermatogenesis of man. Columbia University Press NY.P Oxford & NY. Kiev. 27. ^ Painter T. ^ Stebbins G. 1973.A. Edinburgh.J. 4. 93. Plant Breed. 1958. 11. 2006. 3.

The spermatogenesis of humans. ^ A preparation which includes the dyes Methylene Blue. Bioessays23: 242–250. Chromatin diminution in nematodes.C. 1979. ^ Goday C. Human and mammalian cytogenetics: a historical perspective. and Esteban M.R. Evolutionary genetics.^ a b Gustashaw K.http://www. 2nd ed. Kinetophore reproduction theory may explain rapid chromosome evolution. Chromosomal evolution in higher plants. p218-9 20. 1998. Springer-Verlag. Oxford. ^ Stebbins G. J. ^ Maynard Smith J. . Zoology 37. 2001. 13. 1956. Barch. Exp. Arnold.H & Levan A. 1991. Chromosome stains. 21. ^ Painter T. The Association of Cytogenetic Technologists. 14.S. 15. PNAS 97.J.L. Studies in mammalian spermatogenesis II. 2000. Raven Press. ed. Eosin Y and Azure-A. The chromosome number of man. Hereditas 42. 1996. M.nih.C.B. London. Chromosome elimination in sciarid flies. New York.R. p85-6 18. NY. In The ACT Cytogenetics Laboratory Manual 2nd ed. 9821– 9823. 1971. 1-6. Bioessays18: 133–138.pubmedcentral. and Masters J. ^ Müller F. Bernard V. & Tobler H.C 16. 291-336.gov/articlerender.M.fcgi?artid=34032 19. ^ Tjio J. 1923.12.^ a b Hsu T. 17. ^ Godfrey L.

27.K. Botanical Journal of the Linnean Society 102: 205–217. (1945-05-15). ^ Matthey. Y. 8th ed. Park. ^ Kim.F. 7th ed.A. Ichthyological Research 52 (1): 97. Retrieved 2008-03-18. . C. A dictionary of genetics. Developmental biology.H.K. Oxford U.D. D. Sinauer Associates. ^ Gilbert S. and Benirschke K. 291: 310–16.S. and Mulligan P. Experientia (Basel) 1 (2): 50– 56. "Karyotype of North American shortnose sturgeon Acipenser brevirostrum with the highest chromosome number in the 94– Acipenseriformes" (PDF). ^ Wurster D. 2006. Stamford CT. 1970. doi:10. 26..K. "L'evolution de la formule chromosomiale chez les vertébrés". & Gregory T. Science 168.. 28.doi:10.1007/BF02153623. Muntiacus muntjak: a deer with a low diploid number. Zool. 1990.A.22. Chapman. Nam. 23. F. J.. ^ Wyngaard G.C. ^ Khandelwal S. Noh.. ^ King R. 2001. Indian Muntjac.H. Chapter 9 24. 1364-1366. (2005). Temporal control of DNA replication and the adaptive value of chromatin diminution in copepods. Stansfield W. Exp.1007/s10228-004-0257-z. R.P Oxford & NY. 2006.R. Chromosome evolution in the genus Ophioglossum L. J. 25.. Retrieved 2011-03-16.