ABSTRACT

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.

CHAPTER 1

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

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

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

so that each daughter cell inherits one set of chromatids. and they form the classic four arm structure. During mitosis. Once the cells have divided. This compact form makes the individual chromosomes visible. microtubules grow from centrosomes located at opposite ends of the cell and also attach to the centromere at specialized structures called kinetochores. The microtubules then pull the chromatids apart toward the centrosomes. the chromatin strands become more and more condensed. chromosomes are structurally highly condensed. which enables these giant DNA structures to be contained within a cell nucleus. small) and the longer arms are called q arms (q follows p in the Latin alphabet. This is the only natural context in which individual chromosomes are visible with an optical microscope. q-g "grande"). 1.4 Metaphase chromatin and division In the early stages of mitosis or meiosis (cell division). In spite of their appearance. Such a failure of the separation of homologous chromosomes or sister chromatids is called nondisjunction.1 chromosome paired model Aneuploidy is usually caused by spindle fiber failure in meiosis I or II. the chromatids are uncoiled and DNA can again be transcribed. one of which is present on each sister chromatid. . The shorter arms are called p arms (from the French petit. A special DNA base sequence in the region of the kinetochores provides. They cease to function as accessible genetic material (transcription stops) and become a compact transportable form. along with special proteins. longer-lasting attachment in this region.Fig 1. a pair of sister chromatids attached to each other at the centromere.

.5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones. On average. [1] Bone marrow is also a key component of the lymphatic system. 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.1.7 lbs).6 kg (5. bone marrow accounts for approximately 2. Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle—the division of the mother cell into two daughter cells. organelles and cell membrane into two cells containing roughly equal shares of these cellular components. In humans. It is generally followed immediately by cytokinesis. producing the lymphocytes that support the body's immune system CHAPTER 2 2. The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day. in two separate nuclei. bone marrow in large bones produces new blood cells. bone marrow constitutes 4% of the total body mass of humans. cytoplasm. which divides the nuclei. in adults weighing 65 kg (143 lbs).

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

The sister chromatids are held together by a specialized region of the chromosome known as the centromere. . the period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs. This occurs during the S phase of interphase. 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. and together the two are called sister chromatids. The genome is composed of a number of chromosomes—complexes of tightly-coiled DNA that contain genetic information vital for proper cell function. Because each resultant daughter cell should be genetically identical to the parent cell. Each chromosome now has an identical copy of itself. the parent cell must make a copy of each chromosome before mitosis.

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

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

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

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

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

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

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

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

g.5.5. The end of cytokinesis marks the end of the M-phase. which move along microtubules to the middle of the cell.3 Cell replacement In some parts of body.1Significance Mitosis is important for the maintenance of the chromosomal set. skin and digestive tract.5. zygote and also the basis of the growth of a multicellular body.e.4 Regeneration . This is the basis of the development of a multicellular body from a single cell i.. Following are the occasions in the lives of organism where mitosis happens: 2. New cells are formed by mitosis and so are exact copies of the cells being replaced. 2. Each daughter cell has a complete copy of the genome of its parent cell. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell. e. 2. Similarly. The phragmoplast is a microtubule structure typical for higher plants.division is also driven by vesicles derived from the Golgi apparatus.2 Development and growth The number of cells within an organism increases by mitosis. whereas some green algae use a phycoplast microtubule array during cytokinesis. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis. separating the two nuclei.5. 2. cells are constantly sloughed off and replaced by new ones.

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

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

Metaphase accounts for approximately 4% of the cell cycle's duration.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. Early events of metaphase can . 2. from the ancient Greek(between) and (stage). align in the middle of the cell before being separated into each of the two daughter cells. carrying genetic information. Preceded by events in prometaphase and followed by anaphase. an imaginary line that is equidistant from the two centrosome poles. microtubules formed in prophase have already found and attached themselves to kinetochores in metaphase. An example of a cell that goes through endomitosis is the megakaryocyte.7 Metaphase Metaphase. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. only roughly lining up along the middleline. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. In certain types of cells. The centromeres of the chromosomes convene themselves on the metaphase plate (or equatorial plate).2. is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes. analogous to a tug of war between equally strong people. resulting in cells with many copies of the same chromosome occupying a single nucleus.

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

such as bcr-abl in chronic myelogenous leukemia. .methods like FISH and as a hybridization matrix for comparative genomic hybridization (CGH) experiments. Inspection of the stained metaphase chromosomes allows the determination of numerical and structural changes in the tumor cell genome. for example. 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. losses of chromosomal segments or translocations.

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

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

Rather interestingly.2. is applied after cells have been arrested during cell division by a solution of colchicine.2 Observations on karyotypes 3. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. a suitable dye. The sex of an unborn fetus can be determined by observation of interphase cells. 3.2. For humans. It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes.2 Observations Six different characteristics of karyotypes are usually observed and compared: . the great apes have 48 chromosomes. Usually. Human chromosome 2 was formed by a merger of ancestral chromosomes. Cutting up a photomicrograph and arranging the result into an indisputable karyogram.1 Staining The study of karyotypes is made possible by staining. Arresting mitosis in metaphase by a solution of colchicines 4.3. reducing the number. [16] Sometimes observations may be made on non-dividing (interphase) cells. such as Giemsa. 3. white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture.

3. type. shape and banding of the chromosomes. 6. .1. Differences in number and position of satellites. Differences in degree and distribution of heterochromatic regions. Differences in relative size of chromosomes can only be caused by segmental interchange of unequal lengths. Differences in the position of centromeres. Heterochromatin stains darker than euchromatin. 4. This is brought about by translocations. indicating tighter packing. Differences in absolute sizes of chromosomes. 2. 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 basic number of chromosomes may occur due to successive unequal translocations which finally remove all the essential genetic material from a chromosome. Humans have one pair fewer chromosomes than the great apes. 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. faba chromosomes are many times larger. and mainly consists of genetically inactive repetitive DNA sequences. as well as other cytogenetic information. both have six pairs of chromosomes (n=6) yet V. 5. but the genes have been mostly translocated (added) to other chromosomes. This feature probably reflects different amounts of DNA duplication.

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

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.. In some species. Chromosome elimination. portions of the chromosomes are cast away in particular cells. some organisms go in for large-scale elimination of heterochromatin. In this process.1 Changes during development Instead of the usual gene repression. the general significance of karyotype evolution is obscure. or other kinds of visible adjustment to the karyotype. which were previously inexplicable... despite their construction from the same macromolecules. This variation provides the basis for a range of studies in evolutionary cytology. entire chromosomes are eliminated during development. In A. Although much is known about karyotypes at the descriptive level. as in many sciarid flies. used in conjunction with other phylogenetic data. Godfrey and Masters conclude: "In our view. karyotypic fissioning may help to explain dramatic differences in diploid numbers between closely related species.detailed organization.. Chromatin diminution (founding father: Theodor Boveri). and it is clear that changes in karyotype organization have had effects on the evolutionary course of many species. But. In a review. found in some copepods and roundworms such as Ascaris suum. . "We have a very poor understanding of the causes of karyotype evolution. it is unlikely that one process or the other can independently account for the wide range of karyotype structures that are observed. 3.3. despite many careful investigations. it is quite unclear what the general significance might be.

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

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

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

and 7. 3. In about 6.6 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. that the two chromosome morphs are adapted to different habitats. the great apes have 24x2 chromosomes whereas humans have 23x2.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. the chromosome number is variable from one individual to another. 6. where every number from x = 3 to x = 15 is represented by at least one species. reducing the number. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world. where the gametic (= haploid) numbers form the series x = 3. and Crocus. Classic examples in plants are the genus Crepis. Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups. There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast. 4.500 sq mi (17. 5. [41] Closer to home.000 km2). Well-researched examples are the ladybird beetle Chilocorus stigma.Aneuploidy may also occur within a group of closely related species. the European shrew Sorex araneus. Human chromosome 2 was formed by a merger of ancestral chromosomes. living from rainforests to . some mantids of the genus Ameles. 3. When this happens.

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

It yields a series of lightly and darkly stained bands . so it stains centromeres. adaptive radiations. early-replicating and GC rich. 3. The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions). R-banding is the reverse of G-banding (the R stands for "reverse"). • • C-banding: Giemsa binds to constitutive heterochromatin.7.1 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.the dark regions tend to be heterochromatic.7 Depiction of karyotypes 3. This method will normally produce 300-400 bands in a normal. human genome. late-replicating and AT rich. if less spectacular. . • T-banding: visualize telomeres. The pattern of bands is very similar to that seen in G-banding. • Q-banding is a fluorescent pattern obtained using quinacrine for staining.There are other animals and plants on the Hawaiian archipelago which have undergone similar. The light regions tend to be euchromatic.

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

chromosome 5.2) 3.XX. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale. 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. Short sequences of DNA from specific loci all over the genome are isolated and enumerated. Image processing software then assigns a pseudo color to each spectrally different combination. 3.2. . The critical region for this syndrome is deletion of 15. which is written as 46.del(5)(p15. Because there are a limited number of spectrally-distinct fluorophores. It is written as 46. 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.5p-. This method is also known as virtual karyotyping. a combinatorial labeling method is used to generate many different colors. allowing the visualization of the individually colored chromosomes.7.XX.

CHAPTER 4 .

1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical. Down syndrome. Also documented are trisomy 8.4. X0). XXY is caused by an extra X chromosome. trisomies. in which three copies of a chromosome are present instead of the usual two. otherwise known as 47. • • Patau syndrome is caused by trisomy of chromosome 13. X or 45. Numerical abnormalities. trisomy 9 and trisomy 16. although they generally do not survive to birth. as in derivative chromosome. Some disorders arise from loss of just a piece of one chromosome. also known as aneuploidy. or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells. Structural abnormalities often arise from errors in homologous recombination. translocations. large-scale deletions or duplications. a common chromosomal disease. is caused by trisomy of chromosome 21. Klinefelter syndrome. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body. as in the presence of extra or missing chromosomes. • • Edwards syndrome is caused by trisomy (three copies) of chromosome 18. or structural. the most common male chromosomal disease. inversions. are common numerical abnormalities. including . often occur as a result of nondisjunction during meiosis in the formation of a gamete. Chromosomal abnormalities that lead to disease in humans include • • Turner syndrome results from a single X chromosome (45.

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

There are two main types of translocations. segments from two different chromosomes have been exchanged. Known disorders in humans include Wolf-Hirschhorn syndrome. 4.4. • • Translocations: When a portion of one chromosome is transferred to another 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.2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes). an entire chromosome has . resulting in extra genetic material. Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome. 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. In humans an example of a condition caused by a numerical anomaly is Down Syndrome. Duplications: A portion of the chromosome is duplicated.3 Structural abnormalities When the chromosome's structure is altered. which is caused by partial deletion of the short arm of chromosome 4.). 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. Tetrasomy. In a Robertsonian translocation. and Jacobsen syndrome. also called the terminal 11q deletion disorder. etc. an X. rather than two).

in humans these only occur with chromosomes 13. therefore the genetic material is inverted. 4. can happen after conception.attached to another at the Centromere . • Inversions: A portion of the chromosome has broken off. especially the chromosomes. 4. Chromosome instability syndromes are a group of disorders characterized by chromosomal instability and breakage. however. Chromosome anomalies can be inherited from a parent or be "de novo". and are therefore initially not inherited. They often lead to an increased tendency to develop certain types of malignancies. Therefore. resulting in Mosaicism (where some cells have the anomaly and some do not). the anomaly is present in every cell of the body. turned upside down and reattached. other cytogenetic banding techniques. 14. Some anomalies.4 Cytogenetics Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell. It includes routine analysis of G-Banded chromosomes. 21 and 22.3 Inheritance Most chromosome abnormalities occur as an accident in the egg or sperm. 15. This can happen with or without loss of genetic material. • Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere. This is why chromosome studies are often performed on parents when a child is found to have an anomaly. • Rings: A portion of a chromosome has broken off and formed a circle or ring. as well .

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

reducing the number. 4. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize. McClintock discovered transposons. 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. the great apes have 48 chromosomes.6 Applications in biology 4.6. a find which eventually led to her Nobel Prize in 1983.3. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D.2 Natural populations of Drosophila In the 1930s.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes. Human chromosome 2 was formed by a merger of ancestral chromosomes. Rather interestingly. 4. Arresting mitosis in metaphase by a solution of colchicine 4. persimilis from wild populations in California and neighboring states. In 1931. During her cytogenetic work.6. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes). Using Painter's technique they studied the polytene .

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

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

Techniques were expanded to allow for culture of free amniocytes recovered from amniotic fluid. This allows chromosomes of otherwise equal size to be differentiated as well as to elucidate the breakpoints and constituent chromosomes involved in chromosome translocations. 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.FIG Advent of banding techniques In the late 1960s.8 Beginnings of molecular cytogenetics . and elongation techniques for all culture types that allow for higher resolution banding. 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. 4. Diagrams identifying the chromosomes based on the banding patterns are known as cytogenetic maps.

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

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,

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

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

To compare chromosomes from a band pattern point of view. 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 . 6. 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). To compensate for this inhomogeneity. Therefore. performed by using the algorithm is needed to obtain chromosomes with vertical medial axis. the spatially scaled images are histogram equalized.1) Chromosome extraction—Each chromosome is isolated from the unordered karyogram. 2) Geometrical compensation—The geometric compensation. 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. geometrical and dimensional differences must be removed.

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

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

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

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

19. n1(x1.39.49. for i=1:sx x1=rc(i. y1=rc(i.40.56.imshow(n1.66].j). Test_number=[3.38. Index=Index+1.c] = find(L==L_number((Test_number(x)))). for x=1:46 [r.45.22. n1=zeros(imx. end.65. end flag=0.31.27. [sx sy]=size(rc).[]).52. end %h=figure.42.21.14.29.imy).55.24.10.30.26.59.28.y1)=255.43.50.32.6.60.11.35.51.9.7.41.15. rc = [r c].2).33. end end L_number.end end if flag~=1 L_number(Index)=L(i.8.4.54.57.1).48. 36. .20.62.

Area=zeros(46.'canny'). end end end Circumference(i)=Circumference_sum.Inf).y)==1 Circumference_sum=Circumference_sum+1.1). for i=1:46 f=imread(strcat(num2str(i). Arm_length=zeros(46.'skel'. s1=bwmorph(s. [m n]=size(BW1). Circumference_sum=0. skel=im2double(f).5*graythresh(skel)).end Circumference=zeros(46. Arm_length_sum=0.1. . BW=im2bw(f). BW=double(BW).bmp')). f=imcomplement(f).8). skel=im2bw(skel. BW1=edge(BW. s=bwmorph(skel.'spur'.1).1).'. for x=1:m for y=1:n if BW1(x.

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

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)).2)=i+1.1)=i. Pair(46.2)=j.2). end end Pair. Pair(i. Pair(i. end end end for i=1:45 if Pair(i.2)==46 Pair(46. Pair(i.Area. 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)).1)=i.2)=i.1)=46. Pair=zeros(46.

end f1=imread(strcat(num2str(Pair(i. figure_flag=1. delete(figure_flag)=Pair(i.'.2. figure_flag=figure_flag+1.'.bmp')).2)). if figure_flag~=47 subplot(23.2). end end if flag~=1 if figure_flag~=47 subplot(23.2. end flag=0. . imshow(f2). end f2=imread(strcat(num2str(Pair(i.bmp')).1). figure_flag=figure_flag+1.delete=zeros(46. for i=1:46 for j=1:46 if Pair(i. flag=0.figure_flag).figure_flag).1)==delete(j) flag=1.1)). imshow(f1).

Copenhagen. such as. 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. and Philadelphia. 2) feature extraction from the processed images . a newmetric is proposed to measure the distance between chromosomes to be used in the automatic chromosome pairing procedure. in the scope of karyotyping process used in cytogentic analysis. The algorithm is composed by four main steps: 1) image processing of the karyograms provided by the technicians. based on the MI. plus a new one.end CONCLUTION In this paper. dimensions and banding profiles. The proposed algorithm is based on the traditional features extracted from the karyogram. 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 novel metric distance is proposed to be used in the pairing procedure that linearly combines the distances associated with each feature. shape and band pattern. are processed in order to compensate for geometrical and intensity distortions. 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 . 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). and to normalize their dimensions. 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. In the image processing step.characterizing the size. from the chromosomes in the training set.10% mean classification rate. 3) training of a classifier (performed once) where similarity among chromosomes are characterized. shape. and finally. This normalization is needed to make it possible the band pattern comparison between chromosomes. achieves a 70. 4) pairing. Tests using 19 karyograms based on bone marrow cells. The coefficients of the linear combination are obtained through a training step using chromosomes paired manually by experts.working within an 8-D feature space. The features extracted from the processed images discriminate each pair with respect to their size. 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. The training process consists in the estimation of each vector of coefficient . and band pattern. the romosome images. extracted from the unordered karyogram.working with 22 classes of chromosomes and a LOOCV strategy allowus to conclude that the proposed pairing algorithms.

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

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