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

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

copies of chromosome 21. 1. The simplest gonophores are found in viruses: these DNA or RNA molecules are short linear or circular gonophores that often lack structural proteins. The individual would have Down Syndrome and his/her karyotype would be written 47.3 MUTATIONS IN CHROMOSOME NUMBER Normally. in which an individual has 3.are also found in mitochondria and chloroplasts. the individual carrying the mutation is said to be aneuploid. rather than 2. reflecting their bacterial origins. members of the same species have the same numbers of types of chromosomes (with the exception of sex chromosomes in males and females if sex is chromosomally determined). XX (female) or 46 XY (male).XX. a extra copy of human chromosome 21).g. .+21. If the mutation involves only one or a few chromosomes in the genome (e. Such individuals are called euploid and have the wild-type chromosome complement for the species.+21. Euploid human karyotypes are 46.XY or 47. An example of aneuploidy is trisomy 21. 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.

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

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

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

the period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs. and together the two are called sister chromatids. This occurs during the S phase of interphase. Because each resultant daughter cell should be genetically identical to the parent cell. Each chromosome now has an identical copy of itself. 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. . These two cells are identical and do not differ in any way from the original parent cell.Fig 3 Mitosis cell division The primary result of mitosis is the transferring of the parent cell's genome into two daughter cells. the parent cell must make a copy of each chromosome before mitosis.

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

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

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

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

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

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

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

elongating the cell even more. pinching off the separated nuclei. It "cleans up" the after effects of mitosis. unfold back into chromatin. 2.5 Cytokinesis Cilliate undergoing cytokinesis. forms around each set of separated sister chromosomes. Corresponding sister chromosomes attach at opposite ends of the cell. cell . however. 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. but rather a separate process. Both sets of chromosomes. a cleavage furrow (pinch) containing a contractile ring develops where the metaphase plate used to be. In both animal and plant cells. In animal cells. Mitosis is complete. A new nuclear envelope.2. Cytokinesis is technically not even a phase of mitosis. necessary for completing cell division. the nonkinetochore microtubules continue to lengthen. At telophase. now surrounded by new nuclei. using fragments of the parent cell's nuclear membrane. cytokinesis is a separate process that begins at the same time as telophase.5 Telophase Telophase (from the Greek meaning "end") is a reversal of prophase and prometaphase events.

cells are constantly sloughed off and replaced by new ones.g. The end of cytokinesis marks the end of the M-phase. 2. Following are the occasions in the lives of organism where mitosis happens: 2. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall. e.5.1Significance Mitosis is important for the maintenance of the chromosomal set. Similarly. zygote and also the basis of the growth of a multicellular body. New cells are formed by mitosis and so are exact copies of the cells being replaced. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis.5. Each daughter cell has a complete copy of the genome of its parent cell. The phragmoplast is a microtubule structure typical for higher plants. This is the basis of the development of a multicellular body from a single cell i. whereas some green algae use a phycoplast microtubule array during cytokinesis. which move along microtubules to the middle of the cell.5. separating the two nuclei.5.e. 2. skin and digestive tract. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell.3 Cell replacement In some parts of body.division is also driven by vesicles derived from the Golgi apparatus. 2.4 Regeneration .2 Development and growth The number of cells within an organism increases by mitosis..

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

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

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

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

such as bcr-abl in chronic myelogenous leukemia. . losses of chromosomal segments or translocations. 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.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.

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

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

Human chromosome 2 was formed by a merger of ancestral chromosomes.2 Observations on karyotypes 3. reducing the number.2. [16] Sometimes observations may be made on non-dividing (interphase) cells. such as Giemsa. a suitable dye. 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. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. 3.1 Staining The study of karyotypes is made possible by staining. white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture.2. the great apes have 48 chromosomes. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. Rather interestingly.3.2 Observations Six different characteristics of karyotypes are usually observed and compared: . is applied after cells have been arrested during cell division by a solution of colchicine. The sex of an unborn fetus can be determined by observation of interphase cells. 3. Usually. For humans.

Differences in absolute sizes of chromosomes. A full account of a karyotype may therefore include the number. which (when they occur) are small bodies attached to a chromosome by a thin thread. 6. 4. 2.1. type. both have six pairs of chromosomes (n=6) yet V. This feature probably reflects different amounts of DNA duplication. as well as other cytogenetic information. Humans have one pair fewer chromosomes than the great apes. permitting its loss without penalty to the organism (the dislocation hypothesis). This is brought about by translocations. faba chromosomes are many times larger. Heterochromatin stains darker than euchromatin. 5. Differences in number and position of satellites. 3. Differences in degree and distribution of heterochromatic regions. and mainly consists of genetically inactive repetitive DNA sequences. indicating tighter packing. Differences in relative size of chromosomes can only be caused by segmental interchange of unequal lengths. 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 the position of centromeres. but the genes have been mostly translocated (added) to other chromosomes. . shape and banding of the chromosomes. Differences in basic number of chromosomes may occur due to successive unequal translocations which finally remove all the essential genetic material from a chromosome.

Variation is often found: 1. XY. Normal karyotypes for females contain two X chromosomes and are denoted 46. the same cannot be said for their karyotypes. Between the germ-line and soma (between gametes and the rest of the body) 3.3 The human karyotype Most (but not all) species have a standard karyotype. Fig Diversity and evolution of karyotype Although the replication and transcription of DNA is highly standardized in eukaryotes. which are highly variable. Between members of a population (chromosome polymorphism) 4. There is variation between species in chromosome number. Any variation from the standard karyotype may lead to developmental abnormalities. XX. 3. and in . males have both an X and a Y chromosome denoted 46. The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. Geographical variation between races 5. Between the sexes 2. Mosaics or otherwise abnormal individuals.

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

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

means that chromosome number can vary even within one interbreeding population. where one sex is diploid. and aneuploids are another example. Polyploidy in animals is much less common. of a karyotype is the number of visible major chromosomal arms per set of chromosomes. Polyploidy in lower plants (ferns. FN. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%. 21 and 22).Endopolyploidy occurs when in adult differentiated . FN ≤ 2n. where there are more than two sets of homologous chromosomes in the cells.3. 3. Polyploidy. and the other haploid. Thus. 3. about 70%. 15. occurs mainly in plants. It has been of major significance in plant evolution according to Stebbins. due to the presence of five acrocentric chromosome pairs (13.3 Fundamental number The fundamental number. Humans have FN = 82. Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid. the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present. but it has been significant in some groups. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants. but in grasses the average is much higher. horsetails and psilotales) is also common. 14.4 Ploidy Ploidy is the number of complete sets of chromosomes in a cell. Haplo-diploidy. and in some other groups. though in this case they would not be regarded as normal members of the population. It is a common arrangement in the Hymenoptera.

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

500 sq mi (17.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. When this happens. the chromosome number is variable from one individual to another.Aneuploidy may also occur within a group of closely related species. where the gametic (= haploid) numbers form the series x = 3.6 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 Hawaiian Islands have the most diverse collection of drosophilid flies in the world. 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. In about 6. that the two chromosome morphs are adapted to different habitats. Human chromosome 2 was formed by a merger of ancestral chromosomes. living from rainforests to . 3. where every number from x = 3 to x = 15 is represented by at least one species. 5. the great apes have 24x2 chromosomes whereas humans have 23x2. 6. reducing the number. There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast. [41] Closer to home. some mantids of the genus Ameles.000 km2). Well-researched examples are the ladybird beetle Chilocorus stigma. and 7. 3. the European shrew Sorex araneus. 4. and Crocus.

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

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

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

XX. which is written as 46.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale.2) 3. It is written as 46.chromosome 5. 3.2.del(5)(p15.3 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. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores.XX. This method is also known as virtual karyotyping. The critical region for this syndrome is deletion of 15. allowing the visualization of the individually colored chromosomes. Because there are a limited number of spectrally-distinct fluorophores. . Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope. 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.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.7. a combinatorial labeling method is used to generate many different colors.

CHAPTER 4 .

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

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

• • Translocations: When a portion of one chromosome is transferred to another chromosome. also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21. 4. In a reciprocal translocation. Tetrasomy. which is caused by partial deletion of the short arm of chromosome 4. also called the terminal 11q deletion disorder.2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes). Known human disorders include Charcot-Marie-Tooth disease type 1A which may be caused by duplication of the gene encoding peripheral myelin protein 22 (PMP22) on chromosome 17. and occurs when an individual is missing either a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (Trisomy.3 Structural abnormalities When the chromosome's structure is altered. segments from two different chromosomes have been exchanged. In a Robertsonian translocation. There are two main types of translocations. Duplications: A portion of the chromosome is duplicated. resulting in extra genetic material. This can take several forms: • Deletions: A portion of the chromosome is missing or deleted.4. Known disorders in humans include Wolf-Hirschhorn syndrome. an entire chromosome has . rather than two). an X. In humans an example of a condition caused by a numerical anomaly is Down Syndrome. Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome.). etc. and Jacobsen syndrome.

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

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

a find which eventually led to her Nobel Prize in 1983.6. Human chromosome 2 was formed by a merger of ancestral chromosomes. persimilis from wild populations in California and neighboring states. 4.2 Natural populations of Drosophila In the 1930s. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes).1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. Arresting mitosis in metaphase by a solution of colchicine 4. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes. Rather interestingly. Using Painter's technique they studied the polytene . Cutting up a photomicrograph and arranging the result into an indisputable karyogram. In 1931.6 Applications in biology 4. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. 4. McClintock discovered transposons. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. During her cytogenetic work. the great apes have 48 chromosomes.6. reducing the number. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize.3.

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

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

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

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

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,

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

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

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

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

.2.6. Usually we know what type of errors to expect. Cleaning an image corrupted by noise is thus an important area of image restoration. hence we can choose the most appropriate method for reducing the effects. We may use edges to measure the size of objects in an image. to recognize or classify objects. The general Matlab command for finding edges is edge(image. and we shall look at some of the more straightforward of them. via satellite or wireless transmission.parameters. to isolate particular objects from their background. If one of these matrices describes a two-dimensional finite impulse response .B) computes the two-dimensional convolution of matrices A and B.2.5 Edge detection Edges contain some of the most useful information in an image. If an image is being sent electronically from one place to another. caused by external disturbance. .4 Denoising We may define noise to be any degradation in the image signal.'method'.3 Two dimensional convolutions C = conv2(A. There is a large number of edge finding algorithms in existence. we may expect errors to occur in the image signal. and hence the type of noise on the image. or through networked cable. ) Where the parameters available depend on the method used 6. These errors will appear on the image output in different ways depending on the type of disturbance in the signal. 6.

The size of matrices.hrow. imedfilt2(im1.'sobel'). this case is the same as C = conv2(hcol*hrow. minus one.A). nb]+1)/2)..A) C in each dimension is equal to the sum of the corresponding dimensions of the input A is [ma..(FIR) filter.bmp').7). edge(im1. rgb2gray im2bw(im.[3 3]). ..nb].na+nb-1].na] and the size of B is [mb. the other matrix is filtered in two dimensions.'shape') subsection of the two-dimensional convolution. That is. as specified by the shape parameter Algorithms description 1) Read the image and convert into gray 2)Remove noise 3) Background separation 4) Edge detect 5) Separate the pairs MODULE 1 PSEUDO CODE iimread('12345. The indices of the center element of B are defined as floor(([mb C = conv2(hcol. if the size of then the size of C is [ma+mb-1.0. C = conv2(. convolves A first with the vector hcol along the rows and then returns a with the vector hrow along the columns. If hcol is a column vector and hrow is a row vector.

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

2). end end L_number.7.c] = find(L==L_number((Test_number(x)))).30. 36.21.y1)=255. end.41.43.14.11.39.[]). n1=zeros(imx. for i=1:sx x1=rc(i.imshow(n1.49. Test_number=[3.59.65. end flag=0.19.24. [sx sy]=size(rc).1).15.54.27.35. y1=rc(i. .26. rc = [r c]. end %h=figure.62. for x=1:46 [r.60.end end if flag~=1 L_number(Index)=L(i.48.42.50.66].57.45.40.10.22.6.32.33.29.55. Index=Index+1.38.j).28.9.51. n1(x1.imy).20.4.52.56.31.8.

'canny').1.1). Area=zeros(46.y)==1 Circumference_sum=Circumference_sum+1. .1). for i=1:46 f=imread(strcat(num2str(i). end end end Circumference(i)=Circumference_sum. Circumference_sum=0.5*graythresh(skel)).Inf). Arm_length_sum=0. skel=im2double(f). [m n]=size(BW1).'skel'. BW=double(BW).'. s1=bwmorph(s.1).bmp')).8). BW1=edge(BW.end Circumference=zeros(46.'spur'. f=imcomplement(f). BW=im2bw(f). Arm_length=zeros(46. for x=1:m for y=1:n if BW1(x. s=bwmorph(skel. skel=im2bw(skel.

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

2)==46 Pair(46. Pair(i.2). Pair(i. Pair=zeros(46.1)=i. end end end for i=1:45 if Pair(i.2)=j. end end Pair. Pair(i.2)=i. Pair(46. Pair(i.1)=i.2)=i+1. . 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)).1)=46. 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)).Area.

bmp')). imshow(f2). end f2=imread(strcat(num2str(Pair(i. imshow(f1).1)==delete(j) flag=1.figure_flag).2. if figure_flag~=47 subplot(23.figure_flag).'. for i=1:46 for j=1:46 if Pair(i.'.2.delete=zeros(46. flag=0. end f1=imread(strcat(num2str(Pair(i. figure_flag=figure_flag+1. figure_flag=1. delete(figure_flag)=Pair(i.2)). figure_flag=figure_flag+1. . end flag=0. end end if flag~=1 if figure_flag~=47 subplot(23.1).bmp')).2).1)).

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

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

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

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