ABSTRACT

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

CHAPTER 1

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

especially during early cellular divisions in the zygote. a condition known as monosomy. This results in the former cell having three chromosomes containing the same genes (two sisters and a homologue).7 Consequences of errors Although errors in mitosis are rare. The same division happens during asexual reproduction or vegetative propagation in plants. and the latter cell having only one chromosome (the homologous chromosome). 2.6 Asexual reproduction Some organisms produce genetically similar offspring through asexual reproduction. In non-disjunction. Occasionally when cells experience nondisjunction.Some organisms can regenerate their parts of bodies. the hydra reproduces asexually by budding. a condition known as trisomy. Mitotic errors can be especially dangerous to the organism because future offspring from this parent cell will carry the same disorder. For example. resulting in binucleated cells. For example. Mitosis continues in the cells of bud and it grows into a new individual. they fail to complete cell division and retain both nuclei in one cell. These cells are considered aneuploid. One daughter cell will receive both sister chromosomes and the other will receive none. 2. a condition often associated with cancer. sea star regenerates its lost arm through mitosis.5. The cells at the surface of hydra undergo mitosis and form a mass called bud. a chromosome may fail to separate during anaphase. The production of new cells is achieved by mitosis. the process may go wrong. .5.

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

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

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

. such as bcr-abl in chronic myelogenous leukemia. for example. which may lead to chimeric oncogenes. 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. Inspection of the stained metaphase chromosomes allows the determination of numerical and structural changes in the tumor cell genome.

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

Using cells in culture 2. at first favoring 46. in contrast to their genic contents. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. The name was coined by another German anatomist. The subsequent history of the concept can be followed in the works of Darlington and White. 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. Investigation into the human karyotype took many years to settle the most basic question: how many chromosomes does a normal diploid human cell contain? In 1912. He revised his opinion later from 46 to 48. Considering their techniques. in 1882. the discoverer of mitosis. concluding an XX/XO sex determination mechanism. Their behavior in animal (salamander) cells was described by Walther Flemming. von Waldeyer in 1888.3. which swells them and spreads the chromosomes . The next stage took place after the development of genetics in the early 20th century. Pretreating cells in a hypotonic solution.1 History of karyotype studies Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. these results were quite remarkable. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. and he correctly insisted on humans having an XX/XY system. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes.

2. The sex of an unborn fetus can be determined by observation of interphase cells. white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture. It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes. For humans. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. 3. such as Giemsa. is applied after cells have been arrested during cell division by a solution of colchicine. Human chromosome 2 was formed by a merger of ancestral chromosomes. reducing the number.2. [16] Sometimes observations may be made on non-dividing (interphase) cells. Arresting mitosis in metaphase by a solution of colchicines 4. Usually. a suitable dye.2 Observations on karyotypes 3.3. 3.2 Observations Six different characteristics of karyotypes are usually observed and compared: . the great apes have 48 chromosomes. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. Rather interestingly.1 Staining The study of karyotypes is made possible by staining.

which (when they occur) are small bodies attached to a chromosome by a thin thread. 3. Differences in basic number of chromosomes may occur due to successive unequal translocations which finally remove all the essential genetic material from a chromosome. but the genes have been mostly translocated (added) to other chromosomes. A full account of a karyotype may therefore include the number. and mainly consists of genetically inactive repetitive DNA sequences. 4. both have six pairs of chromosomes (n=6) yet V. 2. Differences in relative size of chromosomes can only be caused by segmental interchange of unequal lengths. indicating tighter packing. Differences in the position of centromeres. Differences in absolute sizes of chromosomes. Differences in number and position of satellites. as well as other cytogenetic information. This is brought about by translocations. This feature probably reflects different amounts of DNA duplication. Chromosomes can vary in absolute size by as much as twenty-fold between genera of the same family: Lotus tenuis and Vicia faba (legumes). shape and banding of the chromosomes. 5.1. permitting its loss without penalty to the organism (the dislocation hypothesis). type. faba chromosomes are many times larger. Humans have one pair fewer chromosomes than the great apes. Heterochromatin stains darker than euchromatin. Differences in degree and distribution of heterochromatic regions. 6. .

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

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

Xinactivation.. thus the mammalian female is a mosaic in respect of her X chromosomes. male = 7 chromosomes. all telocentric. the high record would be somewhere amongst the ferns. But when they obtained a couple more specimens they confirmed [their findings]" Hsu p73-4 The number of chromosomes in the karyotype between (relatively) unrelated species is hugely variable. They kept quiet for two or three years because they thought something was wrong with their tissue culture. In placental mammals. Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a mere 372 chromosomes.suum..3. The existence of supernumerary or B chromosomes . In marsupials it is always the paternal X which is inactivated. The inactivation of one X chromosome takes place during the early development of mammals (see Barr body and dosage compensation). When they looked at the karyotype of the closely related Indian muntjac. all the somatic cell precursors undergo chromatin diminution... The diploid number of the Chinese muntjac. was found to be 46. the inactivation is random as between the two Xs. The low record is held by the nematode Parascaris univalens. where the haploid n = 1.2 Number of chromosomes in a set A spectacular example of variability between closely related species is the muntjac. In human females some 15% of somatic cells escape inactivation. Muntiacus muntjak. with the Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes. "They simply could not believe what they saw. 3. which was investigated by Kurt Benirschke and his colleague Doris Wurster. Muntiacus reevesi. they were astonished to find it had female = 6.

3 Fundamental number The fundamental number.Endopolyploidy occurs when in adult differentiated . where one sex is diploid. 21 and 22).means that chromosome number can vary even within one interbreeding population. Polyploidy. of a karyotype is the number of visible major chromosomal arms per set of chromosomes. and in some other groups. Polyploidy in animals is much less common. It is a common arrangement in the Hymenoptera. though in this case they would not be regarded as normal members of the population. occurs mainly in plants. horsetails and psilotales) is also common. Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid. but in grasses the average is much higher. Humans have FN = 82. but it has been significant in some groups. 14. Thus. 15. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%. and aneuploids are another example. Polyploidy in lower plants (ferns. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants. FN ≤ 2n. 3. due to the presence of five acrocentric chromosome pairs (13.3.4 Ploidy Ploidy is the number of complete sets of chromosomes in a cell. FN. Haplo-diploidy. where there are more than two sets of homologous chromosomes in the cells. 3. and the other haploid. It has been of major significance in plant evolution according to Stebbins. about 70%. the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present.

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

5. where every number from x = 3 to x = 15 is represented by at least one species. Classic examples in plants are the genus Crepis. When this happens. and 7. some mantids of the genus Ameles. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world. where the gametic (= haploid) numbers form the series x = 3.Aneuploidy may also occur within a group of closely related species.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast. 3. reducing the number. In about 6. 6. 3. and Crocus.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.500 sq mi (17. the European shrew Sorex araneus. the chromosome number is variable from one individual to another. Well-researched examples are the ladybird beetle Chilocorus stigma. Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups. [41] Closer to home. Human chromosome 2 was formed by a merger of ancestral chromosomes. 4.000 km2). that the two chromosome morphs are adapted to different habitats. living from rainforests to . the great apes have 24x2 chromosomes whereas humans have 23x2.

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

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

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

which is written as 46. a combinatorial labeling method is used to generate many different colors. 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. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores. It is written as 46. . Because there are a limited number of spectrally-distinct fluorophores.del(5)(p15.2) 3.7. This method is also known as virtual karyotyping. Image processing software then assigns a pseudo color to each spectrally different combination. 3.chromosome 5.3 Spectral karyotype (SKY technique) Spectral karyotyping is a molecular cytogenetic technique used to simultaneously visualize all the pairs of chromosomes in an organism in different colors.5p-.XX. The critical region for this syndrome is deletion of 15. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope. allowing the visualization of the individually colored chromosomes.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale.XX. Short sequences of DNA from specific loci all over the genome are isolated and enumerated.2.

CHAPTER 4 .

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

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

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

4. 14. This is why chromosome studies are often performed on parents when a child is found to have an anomaly. turned upside down and reattached. • Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere. other cytogenetic banding techniques. This can happen with or without loss of genetic material. therefore the genetic material is inverted. however. especially the chromosomes. • Rings: A portion of a chromosome has broken off and formed a circle or ring. and are therefore initially not inherited. Some anomalies. Therefore. It includes routine analysis of G-Banded chromosomes. 21 and 22. 15.in humans these only occur with chromosomes 13. Chromosome anomalies can be inherited from a parent or be "de novo". resulting in Mosaicism (where some cells have the anomaly and some do not). • Inversions: A portion of the chromosome has broken off. as well . They often lead to an increased tendency to develop certain types of malignancies.3 Inheritance Most chromosome abnormalities occur as an accident in the egg or sperm.attached to another at the Centromere . can happen after conception. Chromosome instability syndromes are a group of disorders characterized by chromosomal instability and breakage. the anomaly is present in every cell of the body. 4.4 Cytogenetics Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell.

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

During her cytogenetic work. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes). 4.6.6 Applications in biology 4. 4. persimilis from wild populations in California and neighboring states. Rather interestingly. Using Painter's technique they studied the polytene . Cutting up a photomicrograph and arranging the result into an indisputable karyogram.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist.6. In 1931. reducing the number. McClintock discovered transposons. a find which eventually led to her Nobel Prize in 1983. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize. Human chromosome 2 was formed by a merger of ancestral chromosomes.2 Natural populations of Drosophila In the 1930s. the great apes have 48 chromosomes. Arresting mitosis in metaphase by a solution of colchicine 4. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D.3.

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

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

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

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

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

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

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

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

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

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

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

This compensation algorithm is composed of the following main steps: a) chromosome and medial axis segmentation b) axis smoothing c) interpolationalong orthogonal lines to the smoothed medial axis d) border regularization 3) Shape normalization—The features used in the comparison of chromosomes are grouped into two classes: 1) geometric based 2) pattern based (G-banding).2 Concepts used in this phase 1) Image conversion 2) Denoising .1) Chromosome extraction—Each chromosome is isolated from the unordered karyogram. or at least attenuated. the spatially scaled images are histogram equalized. performed by using the algorithm is needed to obtain chromosomes with vertical medial axis. a dimensional scaling is performed before the pattern features is extracted to make all the chromosome with the same size and aspect ratio by interpolating the original images. 6. Therefore. To compensate for this inhomogeneity. To compare chromosomes from a band pattern point of view. 2) Geometrical compensation—The geometric compensation. 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.

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

Cleaning an image corrupted by noise is thus an important area of image restoration. caused by external disturbance. we may expect errors to occur in the image signal. The general Matlab command for finding edges is edge(image. hence we can choose the most appropriate method for reducing the effects. ) 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.5 Edge detection Edges contain some of the most useful information in an image. and we shall look at some of the more straightforward of them. Usually we know what type of errors to expect. . via satellite or wireless transmission. We may use edges to measure the size of objects in an image.3 Two dimensional convolutions C = conv2(A.'method'.2.B) computes the two-dimensional convolution of matrices A and B.2. If one of these matrices describes a two-dimensional finite impulse response . and hence the type of noise on the image. to isolate particular objects from their background.parameters. There is a large number of edge finding algorithms in existence. 6. . If an image is being sent electronically from one place to another. to recognize or classify objects.6.4 Denoising We may define noise to be any degradation in the image signal. or through networked cable.

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

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

55.c] = find(L==L_number((Test_number(x)))).j).10.27.26. rc = [r c].56.14.41.52.62.9.51.57.[]).48.39.imshow(n1.15.end end if flag~=1 L_number(Index)=L(i.30.y1)=255.38.20.65.21.45.42.49.35. end end L_number.8.6. [sx sy]=size(rc).7. n1(x1. 36.50. for x=1:46 [r.24.19. end flag=0.1).43. Test_number=[3.31.40.32. end %h=figure.60. n1=zeros(imx. . y1=rc(i.11.59.66].33.2).4.imy). Index=Index+1. for i=1:sx x1=rc(i.28. end.54.29.22.

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

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

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)). Pair(46.2)==46 Pair(46.2). .2)=i. Pair(i.1)=i. for i=1:45 min=abs(Circumference(i)-Circumference(i+1))+abs(Arm_length(i)Arm_length(i+1))+abs(Area(i)-Area(i+1)). Pair=zeros(46.1)=i.2)=j. Pair(i. Pair(i. end end end for i=1:45 if Pair(i. end end Pair. Pair(i.Area.2)=i+1.1)=46.

. delete(figure_flag)=Pair(i.1)==delete(j) flag=1. end end if flag~=1 if figure_flag~=47 subplot(23. if figure_flag~=47 subplot(23.delete=zeros(46.1).figure_flag).1)). end flag=0.2)). imshow(f1). figure_flag=1. figure_flag=figure_flag+1.2).figure_flag).bmp')). end f1=imread(strcat(num2str(Pair(i.2.2. imshow(f2).bmp')).'. figure_flag=figure_flag+1. flag=0. for i=1:46 for j=1:46 if Pair(i.'. end f2=imread(strcat(num2str(Pair(i.

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

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

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

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