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

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

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

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

producing the lymphocytes that support the body's immune system CHAPTER 2 2. in adults weighing 65 kg (143 lbs).1 MITOSIS Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets.1. in two separate nuclei. bone marrow constitutes 4% of the total body mass of humans.6 kg (5. On average. bone marrow accounts for approximately 2. which divides the nuclei. In humans. organelles and cell membrane into two cells containing roughly equal shares of these cellular components. . bone marrow in large bones produces new blood cells. It is generally followed immediately by cytokinesis. The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day.5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones. [1] Bone marrow is also a key component of the lymphatic system.7 lbs). cytoplasm. which use the bone marrow vasculature as a conduit to the body's systemic circulation. Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle—the division of the mother cell into two daughter cells.

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

and together the two are called sister chromatids. Because each resultant daughter cell should be genetically identical to the parent cell. . 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. The sister chromatids are held together by a specialized region of the chromosome known as the centromere. 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. the period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs. Each chromosome now has an identical copy of itself.Fig 3 Mitosis cell division The primary result of mitosis is the transferring of the parent cell's genome into two daughter cells.

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

2. grows more and prepares for mitosis (G 2). chromosomes are replicated only during the S phase. a transverse sheet of cytoplasm that bisects the cell along the future plane of cell . Interphase is divided into three phases: G1 (first gap). and finally it divides (M) before restarting the cycle. continues to grow as it duplicates its chromosomes (S). Thus. S (synthesis).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. However. 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. and G2 (second gap). the nucleus has to migrate into the center of the cell before mitosis can begin. It alternates with the much longer interphase.2. In highly vacuolated plant cells. prophase is preceded by a pre-prophase stage. This is achieved through the formation of a phragmosome. 2.1 Preprophase In plant cells only. a cell grows (G1). mainly via proteins. where the cell prepares itself for cell division. All these phases in the interphase are highly regulated. During all three phases. the cell grows by producing proteins and cytoplasmic organelles.

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

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

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

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

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

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

Following are the occasions in the lives of organism where mitosis happens: 2. 2. whereas some green algae use a phycoplast microtubule array during cytokinesis.2 Development and growth The number of cells within an organism increases by mitosis. This is the basis of the development of a multicellular body from a single cell i. New cells are formed by mitosis and so are exact copies of the cells being replaced. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall. Similarly. The phragmoplast is a microtubule structure typical for higher plants. separating the two nuclei. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis.3 Cell replacement In some parts of body. 2.g. 2.5.division is also driven by vesicles derived from the Golgi apparatus.5. The end of cytokinesis marks the end of the M-phase.5. e. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell.. cells are constantly sloughed off and replaced by new ones.e.5. which move along microtubules to the middle of the cell.4 Regeneration .1Significance Mitosis is important for the maintenance of the chromosomal set. zygote and also the basis of the growth of a multicellular body. skin and digestive tract. Each daughter cell has a complete copy of the genome of its parent cell.

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

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

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

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

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

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

which swells them and spreads the chromosomes . concluding an XX/XO sex determination mechanism. New techniques were needed to definitively solve the problem: 1. Painter in 1922 was not certain whether the diploid number of humans was 46 or 48.3. 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. these results were quite remarkable. The next stage took place after the development of genetics in the early 20th century. Using cells in culture 2. at first favoring 46.1 History of karyotype studies Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. The subsequent history of the concept can be followed in the works of Darlington and White. Pretreating cells in a hypotonic solution. He revised his opinion later from 46 to 48. Considering their techniques. von Waldeyer in 1888. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. Their behavior in animal (salamander) cells was described by Walther Flemming. the discoverer of mitosis. in 1882. 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 name was coined by another German anatomist. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes.

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

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

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

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

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

means that chromosome number can vary even within one interbreeding population. occurs mainly in plants.3 Fundamental number The fundamental number. and aneuploids are another example. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants. 3. 21 and 22). horsetails and psilotales) is also common. Thus. FN ≤ 2n. about 70%.Endopolyploidy occurs when in adult differentiated . 3. Haplo-diploidy. It is a common arrangement in the Hymenoptera. Humans have FN = 82. 15. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%. Polyploidy in lower plants (ferns. 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. but it has been significant in some groups. and in some other groups. of a karyotype is the number of visible major chromosomal arms per set of chromosomes.4 Ploidy Ploidy is the number of complete sets of chromosomes in a cell. Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid. FN. 14.3. Polyploidy. though in this case they would not be regarded as normal members of the population. and the other haploid. due to the presence of five acrocentric chromosome pairs (13. the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present. but in grasses the average is much higher. where one sex is diploid. Polyploidy in animals is much less common.

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

5. [41] Closer to home. that the two chromosome morphs are adapted to different habitats. reducing the number. Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups. the European shrew Sorex araneus. and Crocus. living from rainforests to .6 Species trees The detailed study of chromosome banding in insects with polytene chromosomes can reveal relationships between closely related species: the classic example is the study of chromosome banding in Hawaiian drosophilids by Hampton Carson. the great apes have 24x2 chromosomes whereas humans have 23x2. and 7. When this happens. 3. Human chromosome 2 was formed by a merger of ancestral chromosomes. In about 6. where the gametic (= haploid) numbers form the series x = 3. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world. There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast. 3.000 km2).5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. 4. Classic examples in plants are the genus Crepis.Aneuploidy may also occur within a group of closely related species. some mantids of the genus Ameles. Well-researched examples are the ladybird beetle Chilocorus stigma. 6.500 sq mi (17. where every number from x = 3 to x = 15 is represented by at least one species. the chromosome number is variable from one individual to another.

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

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

a dye. In the "classic" (depicted) karyotype. less frequently Quinacrine. often Giemsa (G-banding). For example. respectively. Giemsa is specific for the phosphate groups of DNA. Each chromosome has a characteristic banding pattern that helps to identify them.• Silver staining: Silver nitrate stains the nucleolar organization regionassociated protein. is used to stain bands on the chromosomes. and the long arm on the bottom. Cri du chat syndrome involves a deletion on the short arm of . 3.7. both chromosomes in a pair will have the same banding pattern. Quinacrine binds to the adeninethymine-rich regions. This yields a dark region where the silver is deposited. In addition. Karyotypes are arranged with the short arm of the chromosome on top. denoting the activity of rRNA genes within the NOR. the differently stained regions and sub-regions are given numerical designations from proximal to distal on the chromosome arms.2 Classic karyotype cytogenetics Karyogram from a human female lymphocyte probed for the Alu sequence using FISH. Some karyotypes call the short and long arms p and q.

del(5)(p15.7.chromosome 5. allowing the visualization of the individually colored chromosomes.XX.XX.2) 3. This method is also known as virtual karyotyping. Image processing software then assigns a pseudo color to each spectrally different combination. .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.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale. Because there are a limited number of spectrally-distinct fluorophores. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores. a combinatorial labeling method is used to generate many different colors. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope.5p-. 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. 3. It is written as 46. The critical region for this syndrome is deletion of 15.2. Short sequences of DNA from specific loci all over the genome are isolated and enumerated. which is written as 46.

CHAPTER 4 .

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

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

an entire chromosome has .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. Duplications: A portion of the chromosome is duplicated. Tetrasomy. There are two main types of translocations. rather than two). 4. segments from two different chromosomes have been exchanged.3 Structural abnormalities When the chromosome's structure is altered. which is caused by partial deletion of the short arm of chromosome 4. and Jacobsen syndrome. In a Robertsonian translocation. In a reciprocal translocation. Known human disorders include Charcot-Marie-Tooth disease type 1A which may be caused by duplication of the gene encoding peripheral myelin protein 22 (PMP22) on chromosome 17. etc. This can take several forms: • Deletions: A portion of the chromosome is missing or deleted.4. In humans an example of a condition caused by a numerical anomaly is Down Syndrome. an X. Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome. resulting in extra genetic material. Known disorders in humans include Wolf-Hirschhorn syndrome.). also called the terminal 11q deletion disorder. • • 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.

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

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

1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist.6 Applications in biology 4. 4. a find which eventually led to her Nobel Prize in 1983. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. 4. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. McClintock discovered transposons. reducing the number. Rather interestingly. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. Arresting mitosis in metaphase by a solution of colchicine 4. During her cytogenetic work. persimilis from wild populations in California and neighboring states. Human chromosome 2 was formed by a merger of ancestral chromosomes.2 Natural populations of Drosophila In the 1930s.6.6. 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 . In 1931. the great apes have 48 chromosomes. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize.3.

such as Down's syndrome. This had the benefit of eliminating migration as a possible explanation of the results. 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. 4.7 Human abnormalities and medical applications In the event of procedures which allowed easy enumeration of chromosomes. as they would if selectively neutral. discoveries were quickly made related to aberrant chromosomes or chromosome number. 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. as with most polymorphisms. It was found that the various chromosome types do not fluctuate at random. which enabled feeding. Evidence rapidly accumulated to show that natural selection was responsible. Lejeune discovered patients with Down syndrome had an extra copy of chromosome 21. Dobzhansky bred populations in population cages. but adjust to certain frequencies at which they become stabilised. Down syndrome is also referred to as trisomy 21. Stocks containing inversions at a known initial frequency can be maintained in controlled conditions. . All the flies look alike whatever inversions they carry: this is an example of a cryptic polymorphism. cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy. In some congenital disorders. Using a method invented by L'Heretier and Teissier. breeding and sampling whilst preventing escape. In 1959.

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

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

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

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,

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

.MATLAB provides a number of features for documenting and sharing your work. one line of MATLAB code can often replace several lines of C or C++ code. With the MATLAB language. 6. MATLAB eliminates the need for ‘for’ loops. specifying data types. 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. As a result. 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. such as declaring variables. and distribute your MATLAB algorithms and applications. You can integrate your MATLAB code with other languages and applications. The MATLAB language supports the vector and matrix operations that are fundamental to engineering and scientific problems.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. and allocating memory. In many cases. It enables fast development and execution. These effects must be compensated to improve the results of the pairing algorithm.

2 Concepts used in this phase 1) Image conversion 2) Denoising . To compare chromosomes from a band pattern point of view. 2) Geometrical compensation—The geometric compensation. Therefore. the spatially scaled images are histogram equalized. 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. To compensate for this inhomogeneity. 4) Intensity compensation—The metaphase plaque from which the chromosomes are extracted does not present a uniform brightness and contrast. performed by using the algorithm is needed to obtain chromosomes with vertical medial axis. geometrical and dimensional differences must be removed. 6.1) Chromosome extraction—Each chromosome is isolated from the unordered karyogram. 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). or at least attenuated.

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

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

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

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

end. 36.9.60.10.11.57.j). for i=1:sx x1=rc(i.31.48. end flag=0.30.2).imy).22.27.29. Index=Index+1.24.y1)=255. n1=zeros(imx.end end if flag~=1 L_number(Index)=L(i.20.40.21.33.55.59.43. rc = [r c]. for x=1:46 [r.49.32.c] = find(L==L_number((Test_number(x)))). end %h=figure. [sx sy]=size(rc). Test_number=[3.[]).6.8. n1(x1.4.50.38.51.15. . y1=rc(i.7.62. end end L_number.52.54.45.28.56.imshow(n1.42.65.35.41.26.1).14.39.66].19.

end end end Circumference(i)=Circumference_sum.'. Circumference_sum=0. for i=1:46 f=imread(strcat(num2str(i). s1=bwmorph(s. skel=im2bw(skel.y)==1 Circumference_sum=Circumference_sum+1. s=bwmorph(skel. BW=im2bw(f). BW1=edge(BW.'canny'). [m n]=size(BW1).Inf).'skel'. Arm_length_sum=0.1).1. .5*graythresh(skel)).8).bmp')). BW=double(BW).1). for x=1:m for y=1:n if BW1(x. Area=zeros(46. Arm_length=zeros(46.1).end Circumference=zeros(46. f=imcomplement(f). skel=im2double(f).'spur'.

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

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

end f2=imread(strcat(num2str(Pair(i. end end if flag~=1 if figure_flag~=47 subplot(23.2). delete(figure_flag)=Pair(i. end f1=imread(strcat(num2str(Pair(i.delete=zeros(46.2)). end flag=0. if figure_flag~=47 subplot(23.'. .'. imshow(f1).1)). flag=0.bmp')).bmp')). figure_flag=figure_flag+1. imshow(f2).2. for i=1:46 for j=1:46 if Pair(i.figure_flag). figure_flag=1.1)==delete(j) flag=1.1).figure_flag).2. figure_flag=figure_flag+1.

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

3) training of a classifier (performed once) where similarity among chromosomes are characterized. 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. extracted from the unordered karyogram. are processed in order to compensate for geometrical and intensity distortions. Tests using 19 karyograms based on bone marrow cells. The training process consists in the estimation of each vector of coefficient . Here. and finally. shape and band pattern. The coefficients of the linear combination are obtained through a training step using chromosomes paired manually by experts. the romosome images.10% mean classification rate. a novel metric distance is proposed to be used in the pairing procedure that linearly combines the distances associated with each feature. and band pattern. 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 . 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. This normalization is needed to make it possible the band pattern comparison between chromosomes.working with 22 classes of chromosomes and a LOOCV strategy allowus to conclude that the proposed pairing algorithms. In the image processing step.characterizing the size. The features extracted from the processed images discriminate each pair with respect to their size. achieves a 70. shape. from the chromosomes in the training set. and to normalize their dimensions. 4) pairing.working within an 8-D feature space. 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).

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

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