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

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

are also found in mitochondria and chloroplasts, reflecting their bacterial origins. The simplest gonophores are found in viruses: these DNA or RNA molecules are short linear or circular gonophores that often lack structural proteins.

1.3 MUTATIONS IN CHROMOSOME NUMBER Normally, 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). Such individuals are called euploid and have the wild-type chromosome complement for the species. Euploid human karyotypes are 46, XX (female) or 46 XY (male). 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. If the mutation involves only one or a few chromosomes in the genome (e.g. a extra copy of human chromosome 21), the individual carrying the mutation is said to be aneuploid. An example of aneuploidy is trisomy 21, in which an individual has 3, rather than 2, copies of chromosome 21. The individual would have Down Syndrome and his/her karyotype would be written 47,+21,XY or 47,+21,XX.

Fig 1.1 chromosome paired model Aneuploidy is usually caused by spindle fiber failure in meiosis I or II. Such a failure of the separation of homologous chromosomes or sister chromatids is called nondisjunction. 1.4 Metaphase chromatin and division In the early stages of mitosis or meiosis (cell division), the chromatin strands become more and more condensed. They cease to function as accessible genetic material (transcription stops) and become a compact transportable form. This compact form makes the individual chromosomes visible, and they form the classic four arm structure, a pair of sister chromatids attached to each other at the centromere. The shorter arms are called p arms (from the French petit, small) and the longer arms are called q arms (q follows p in the Latin alphabet; q-g "grande"). This is the only natural context in which individual chromosomes are visible with an optical microscope. During mitosis, microtubules grow from centrosomes located at opposite ends of the cell and also attach to the centromere at specialized structures called kinetochores, one of which is present on each sister chromatid. A special DNA base sequence in the region of the kinetochores provides, along with special proteins, longer-lasting attachment in this region. The microtubules then pull the chromatids apart toward the centrosomes, so that each daughter cell inherits one set of chromatids. Once the cells have divided, the chromatids are uncoiled and DNA can again be transcribed. In spite of their appearance, chromosomes are structurally highly condensed, which enables these giant DNA structures to be contained within a cell nucleus.

1.5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones. In humans, bone marrow in large bones produces new blood cells. On average, bone marrow constitutes 4% of the total body mass of humans; in adults weighing 65 kg (143 lbs), bone marrow accounts for approximately 2.6 kg (5.7 lbs). The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day, which use the bone marrow vasculature as a conduit to the body's systemic circulation. [1] Bone marrow is also a key component of the lymphatic system, producing the lymphocytes that support the body's immune system

CHAPTER 2 2.1 MITOSIS Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets, in two separate nuclei. It is generally followed immediately by cytokinesis, which divides the nuclei, cytoplasm, organelles and cell membrane into two cells containing roughly equal shares of these cellular components. Mitosis and cytokinesis together define the mitotic (M) phase of the cell cyclethe division of the mother cell into two daughter cells,

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

Fig 3 Mitosis cell division The primary result of mitosis is the transferring of the parent cell's genome into two daughter cells. 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 chromosomescomplexes of tightly-coiled DNA that contain genetic information vital for proper cell function. Because each resultant daughter cell should be genetically identical to the parent cell, the parent cell must make a copy of each chromosome before mitosis. This occurs during the S phase of interphase, the period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs. Each chromosome now has an identical copy of itself, and together the two are called sister chromatids. The sister chromatids are held together by a specialized region of the chromosome known as the centromere.

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

2.2 Phases of cell cycle and mitosis Interphase

Fig 4 The cell cycle

The mitotic phase is a relatively short period of the cell cycle. It alternates with the much longer interphase, where the cell prepares itself for cell division. Interphase is divided into three phases: G1 (first gap), S (synthesis), and G2 (second gap). During all three phases, the cell grows by producing proteins and cytoplasmic organelles. However, chromosomes are replicated only during the S phase. Thus, a cell grows (G1), continues to grow as it duplicates its chromosomes (S), grows more and prepares for mitosis (G 2), and finally it divides (M) before restarting the cycle. All these phases in the interphase are highly regulated, mainly via proteins. The phases follow one another in strict order and there are "checkpoints" that give the cell the cues to proceed from one phase to another. 2.2.1 Preprophase In plant cells only, prophase is preceded by a pre-prophase stage. In highly vacuolated plant cells, the nucleus has to migrate into the center of the cell before mitosis can begin. This is achieved through the formation of a phragmosome, a transverse sheet of cytoplasm that bisects the cell along the future plane of cell

division. In addition to phragmosome formation, 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. This band marks the position where the cell will eventually divide. The cells of higher plants (such as the flowering plants) lack centrioles; instead, 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. The preprophase band disappears during nuclear envelope disassembly and spindle formation in prometaphase.

Prophase: The two round objects above the nucleus Metaphase: The are the centrosomes. The chromosomes have chromatin has condensed. aligned at the metaphase plate.

Early anaphase: The Prometaphase: The kinetochore nuclear membrane has microtubules shorten. degraded, and microtubules have invaded the nuclear Prophase space. These microtubules can attach to kinetochores or they can interact with opposing microtubules.

Telophase: The decondensing chromosomes are surrounded by nuclear membranes. Cytokinesis has already begun; the pinched area is known as the cleavage furrow.

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

formation of the spindle, since they are absent from plants, and centrosomes are not always used in mitosis. 2.2.2 Prometaphase The nuclear envelope disassembles and microtubules invade the nuclear space. This is called open mitosis, and it occurs in most multicellular organisms. Fungi and some protists, such as algae or trichomonads, undergo a variation called closed mitosis where the spindle forms inside the nucleus, or its microtubules are able to penetrate an intact nuclear envelope. Each chromosome forms two kinetochores at the centromere, one attached at each chromatid. A kinetochore is a complex protein structure that is analogous to a ring for the microtubule hook; it is the point where microtubules attach themselves to the chromosome ( about 1-40 in number, on an average 20 ). Although the kinetochore structure and function are not fully understood, it is known that it contains some form of molecular motor. When a microtubule connects with the kinetochore, the motor activates, using energy from ATP to "crawl" up the tube toward the originating centrosome. This motor activity, coupled with polymerisation and depolymerisation of microtubules, provides the pulling force necessary to later separate the chromosome's two chromatids. When the spindle grows to sufficient length, kinetochore microtubules begin searching for kinetochores to attach to. A number of nonkinetochore microtubules find and interact with corresponding nonkinetochore microtubules from the opposite centrosome to form the mitotic spindle. Prometaphase is sometimes considered part of prophase.

In the fishing pole analogy, the kinetochore would be the "hook" that catches a sister chromatid or "fish". 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.
2.3 Metaphase

A cell in late metaphase. All chromosomes (blue) but one have arrived at the metaphase plate.

Metaphase comes from the Greek meaning "after." Microtubules find and attach to kinetochores in prometaphase. Then the two centrosomes start pulling the chromosomes through their attached centromeres towards the two ends of the cell. As a result, the chromosomes come under longitudinal tension from the two ends of the cell. The centromeres of the chromosomes, in some sense, convene along the metaphase plate or equatorial plane, 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, analogous to a tug-of-war between people of equal strength. In certain types of cells, chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly, only roughly lining up along the midline.

Because proper chromosome separation requires that every kinetochore be attached to a bundle of microtubules (spindle fibres), it is thought that unattached kinetochores generate a signal to prevent premature progression to anaphase without all chromosomes being aligned. The signal creates the mitotic spindle checkpoint. 2.4 Anaphase When every kinetochore is attached to a cluster of microtubules and the chromosomes have lined up along the metaphase plate, the cell proceeds to anaphase (from the Greek meaning up, against, back, or re-). Two events then occur: first, the proteins that bind sister chromatids together are cleaved, allowing them to separate. These sister chromatids, which have now become distinct sister chromosomes, are pulled apart by shortening kinetochore microtubules and move toward the respective centrosomes to which they are attached. Next, the nonkinetochore microtubules elongate, pulling the centrosomes (and the set of chromosomes to which they are attached) apart to opposite ends of the cell. The force that causes the centrosomes to move towards the ends of the cell is still unknown, although there is a theory that suggests that the rapid assembly and breakdown of microtubules may cause this movement. These two stages are sometimes called early and late anaphase. Early anaphase is usually defined as the separation of the sister chromatids, while late anaphase is the elongation of the microtubules and the chromosomes being pulled farther apart. At the end of anaphase, the cell has succeeded in separating identical copies of the genetic material into two distinct populations.

2.5 Telophase Telophase (from the Greek meaning "end") is a reversal of prophase and prometaphase events. It "cleans up" the after effects of mitosis. At telophase, the nonkinetochore microtubules continue to lengthen, elongating the cell even more. Corresponding sister chromosomes attach at opposite ends of the cell. A new nuclear envelope, using fragments of the parent cell's nuclear membrane, forms around each set of separated sister chromosomes. Both sets of chromosomes, now surrounded by new nuclei, unfold back into chromatin. Mitosis is complete, but cell division is not yet complete.
2.5 Cytokinesis

Cilliate undergoing cytokinesis, with the cleavage furrow being clearly visible

Cytokinesis is often mistakenly thought to be the final part of telophase; however, cytokinesis is a separate process that begins at the same time as telophase. Cytokinesis is technically not even a phase of mitosis, but rather a separate process, necessary for completing cell division. In animal cells, a cleavage furrow (pinch) containing a contractile ring develops where the metaphase plate used to be, pinching off the separated nuclei. In both animal and plant cells, cell

division is also driven by vesicles derived from the Golgi apparatus, which move along microtubules to the middle of the cell. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall, separating the two nuclei. The phragmoplast is a microtubule structure typical for higher plants, whereas some green algae use a phycoplast microtubule array during cytokinesis. Each daughter cell has a complete copy of the genome of its parent cell. The end of cytokinesis marks the end of the M-phase. 2.5.1Significance Mitosis is important for the maintenance of the chromosomal set; each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell. Following are the occasions in the lives of organism where mitosis happens: 2.5.2 Development and growth The number of cells within an organism increases by mitosis. This is the basis of the development of a multicellular body from a single cell i.e., zygote and also the basis of the growth of a multicellular body. 2.5.3 Cell replacement In some parts of body, e.g. skin and digestive tract, cells are constantly sloughed off and replaced by new ones. New cells are formed by mitosis and so are exact copies of the cells being replaced. Similarly, RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis. 2.5.4 Regeneration

Some organisms can regenerate their parts of bodies. The production of new cells is achieved by mitosis. For example; sea star regenerates its lost arm through mitosis. 2.5.6 Asexual reproduction Some organisms produce genetically similar offspring through asexual reproduction. For example, the hydra reproduces asexually by budding. 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. The same division happens during asexual reproduction or vegetative propagation in plants. 2.5.7 Consequences of errors Although errors in mitosis are rare, the process may go wrong, especially during early cellular divisions in the zygote. Mitotic errors can be especially dangerous to the organism because future offspring from this parent cell will carry the same disorder. In non-disjunction, a chromosome may fail to separate during anaphase. One daughter cell will receive both sister chromosomes and the other will receive none. This results in the former cell having three chromosomes containing the same genes (two sisters and a homologue), a condition known as trisomy, and the latter cell having only one chromosome (the homologous chromosome), a condition known as monosomy. These cells are considered aneuploid, a condition often associated with cancer. Occasionally when cells experience nondisjunction, they fail to complete cell division and retain both nuclei in one cell, resulting in binucleated cells.

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

2.6 Endomitosis Endomitosis is a variant of mitosis without nuclear or cellular division, 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. An example of a cell that goes through endomitosis is the megakaryocyte.

2.7 Metaphase Metaphase, from the ancient Greek(between) and (stage), is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes, carrying genetic information, align in the middle of the cell before being separated into each of the two daughter cells. Metaphase accounts for approximately 4% of the cell cycle's duration. Preceded by events in prometaphase and followed by anaphase, microtubules formed in prophase have already found and attached themselves to kinetochores in metaphase. The centromeres of the chromosomes convene themselves on the metaphase plate (or equatorial plate), 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, analogous to a tug of war between equally strong people. In certain types of cells, chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly, only roughly lining up along the middleline. Early events of metaphase can

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. One of the cell cycle checkpoints occurs during prometaphase and metaphase. Only after all chromosomes have become aligned at the metaphase plate, when every kinetochore is properly attached to a bundle of microtubules, does the cell enter anaphase. It is thought that unattached or improperly attached kinetochores generate a signal to prevent premature progression to anaphase, even if most of the kinetochores have been attached and most of the chromosomes have been aligned. Such a signal creates the mitotic spindle checkpoint. This would be accomplished by regulation of the anaphase-promoting complex, securin, and separase. 2.8 Metaphase in cytogenetics and cancer studies The analysis of metaphase chromosomes is one of the main tools of classical cytogenetics and cancer studies. Chromosomes are condensed(Thickened) and highly coiled in metaphase, which makes them most suitable for visual analysis. Metaphase chromosomes make the classical picture of chromosomes (karyotype). For classical cytogenetic analyses, cells are grown in short term culture and arrested in metaphase using mitotic inhibitor. 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). Staining of the slides, often with Giemsa (G banding) or Quinacrine, produces a pattern of in total up to several hundred bands. Normal metaphase spreads are used in

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, for example, losses of chromosomal segments or translocations, which may lead to chimeric oncogenes, such as bcr-abl in chronic myelogenous leukemia.

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

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

Pretreating cells in a hypotonic solution, which swells them and spreads the chromosomes

3.

Arresting mitosis in metaphase by a solution of colchicines

4. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes. Rather interestingly, the great apes have 48 chromosomes. Human chromosome 2 was formed by a merger of ancestral chromosomes, reducing the number. 3.2 Observations on karyotypes 3.2.1 Staining The study of karyotypes is made possible by staining. Usually, a suitable dye, such as Giemsa, is applied after cells have been arrested during cell division by a solution of colchicine. For humans, white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture.
[16]

Sometimes observations may be made on non-dividing (interphase) cells. The

sex of an unborn fetus can be determined by observation of interphase cells.

3.2.2 Observations Six different characteristics of karyotypes are usually observed and compared:

1.

Differences in absolute sizes of chromosomes. Chromosomes can vary in absolute size by as much as twenty-fold between genera of the same family: Lotus tenuis and Vicia faba (legumes), both have six pairs of chromosomes (n=6) yet V. faba chromosomes are many times larger. This feature probably reflects different amounts of DNA duplication. Differences in the position of centromeres. This is brought about by translocations.

2.

3. Differences in relative size of chromosomes can only be caused by segmental interchange of unequal lengths. 4. Differences in basic number of chromosomes may occur due to successive unequal translocations which finally remove all the essential genetic material from a chromosome, permitting its loss without penalty to the organism (the dislocation hypothesis). Humans have one pair fewer chromosomes than the great apes, but the genes have been mostly translocated (added) to other chromosomes. 5. Differences in number and position of satellites, which (when they occur) are small bodies attached to a chromosome by a thin thread.
6.

Differences

in

degree

and

distribution

of heterochromatic regions.

Heterochromatin stains darker than euchromatin, indicating tighter packing, and mainly consists of genetically inactive repetitive DNA sequences. A full account of a karyotype may therefore include the number, type, shape and banding of the chromosomes, as well as other cytogenetic information.

Variation is often found: 1. Between the sexes

2.

Between the germ-line and soma (between gametes and the rest of the body)

3. Between members of a population (chromosome polymorphism) 4. Geographical variation between races 5. Mosaics or otherwise abnormal individuals.

3.3 The human karyotype Most (but not all) species have a standard karyotype. The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. Normal karyotypes for females contain two X chromosomes and are denoted 46, XX; males have both an X and a Y chromosome denoted 46, XY. Any variation from the standard karyotype may lead to developmental abnormalities.

Fig Diversity and evolution of karyotype Although the replication and transcription of DNA is highly standardized in eukaryotes, the same cannot be said for their karyotypes, which are highly variable. There is variation between species in chromosome number, and in

detailed organization, despite their construction from the same macromolecules. This variation provides the basis for a range of studies in evolutionary cytology. In some cases there is even significant variation within species. In a review, Godfrey and Masters conclude: "In our view, it is unlikely that one process or the other can independently account for the wide range of karyotype structures that are observed... But, used in conjunction with other phylogenetic data, karyotypic fissioning may help to explain dramatic differences in diploid numbers between closely related species, which were previously inexplicable. Although much is known about karyotypes at the descriptive level, and it is clear that changes in karyotype organization have had effects on the evolutionary course of many species, it is quite unclear what the general significance might be. "We have a very poor understanding of the causes of karyotype evolution; despite many careful investigations... the general significance of karyotype evolution is obscure. 3.3.1 Changes during development Instead of the usual gene repression, some organisms go in for large-scale elimination of heterochromatin, or other kinds of visible adjustment to the karyotype. Chromosome elimination. In some species, as in many sciarid flies, entire chromosomes are eliminated during development. Chromatin diminution (founding father: Theodor Boveri). In this process, found in some copepods and roundworms such as Ascaris suum, portions of the chromosomes are cast away in particular cells. This process is a carefully organised genome rearrangement where new telomeres are constructed and certain heterochromatin regions are lost. In A.

suum, all the somatic cell precursors undergo chromatin diminution. Xinactivation. The inactivation of one X chromosome takes place during the early development of mammals (see Barr body and dosage compensation). In placental mammals, the inactivation is random as between the two Xs; thus the mammalian female is a mosaic in respect of her X chromosomes. In marsupials it is always the paternal X which is inactivated. In human females some 15% of somatic cells escape inactivation. 3.3.2 Number of chromosomes in a set A spectacular example of variability between closely related species is the muntjac, which was investigated by Kurt Benirschke and his colleague Doris Wurster. The diploid number of the Chinese muntjac, Muntiacus reevesi, was found to be 46, all telocentric. When they looked at the karyotype of the closely related Indian muntjac, Muntiacus muntjak, they were astonished to find it had female = 6, male = 7 chromosomes. "They simply could not believe what they saw... They kept quiet for two or three years because they thought something was wrong with their tissue culture... 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 low record is held by the nematode Parascaris univalens, where the haploid n = 1; the high record would be somewhere amongst the ferns, with the Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes. Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a mere 372 chromosomes. The existence of supernumerary or B chromosomes

means that chromosome number can vary even within one interbreeding population; and aneuploids are another example, though in this case they would not be regarded as normal members of the population. 3.3.3 Fundamental number The fundamental number, FN, of a karyotype is the number of visible major chromosomal arms per set of chromosomes. Thus, FN 2n, the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present. Humans have FN = 82, due to the presence of five acrocentric chromosome pairs (13, 14, 15, 21 and 22).
3.4 Ploidy

Ploidy is the number of complete sets of chromosomes in a cell. Polyploidy, where there are more than two sets of homologous chromosomes in the cells, occurs mainly in plants. It has been of major significance in plant evolution according to Stebbins. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%, but in grasses the average is much higher, about 70%. Polyploidy in lower plants (ferns, horsetails and psilotales) is also common, and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants. Polyploidy in animals is much less common, but it has been significant in some groups. Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid. Haplo-diploidy, where one sex is diploid, and the other haploid. It is a common arrangement in the Hymenoptera, and in some other groups.Endopolyploidy occurs when in adult differentiated

tissues the cells have ceased to divide by mitosis, but the nuclei contain more than the original somatic number of chromosomes. 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. In many instances, endopolyploid nuclei contain tens of thousands of chromosomes (which cannot be exactly counted). 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. 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 phenomenon occurs sporadically throughout the eukaryote kingdom from protozoa to man; it is diverse and complex, and serves differentiation and morphogenesis in many ways. See palaeopolyploidy for the investigation of ancient karyotype duplications. 3.5 Aneuploidy Aneuploidy is the condition in which the chromosome number in the cells is not the typical number for the species. This would give rise to a chromosome abnormality such as an extra chromosome or one or more chromosomes lost. Abnormalities in chromosome number usually cause a defect in development. Down syndrome and Turner syndrome are examples of this.

Aneuploidy may also occur within a group of closely related species. Classic examples in plants are the genus Crepis, where the gametic (= haploid) numbers form the series x = 3, 4, 5, 6, and 7; and Crocus, where every number from x = 3 to x = 15 is represented by at least one species. Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups. [41] Closer to home, the great apes have 24x2 chromosomes whereas humans have 23x2. Human chromosome 2 was formed by a merger of ancestral chromosomes, reducing the number. 3.5 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. Well-researched examples are the ladybird beetle Chilocorus stigma, some mantids of the genus Ameles, the European shrew Sorex araneus. There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast, that the two chromosome morphs are adapted to different habitats. 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. In about 6,500 sq mi (17,000 km2), the Hawaiian Islands have the most diverse collection of drosophilid flies in the world, living from rainforests to

subalpine meadows. These roughly 800 Hawaiian drosophilid species are usually assigned to two genera, Drosophila and Scaptomyza, in the family Drosophilidae. The polytene banding of the 'picture wing' group, the best-studied group of Hawaiian drosophilids, enabled Carson to work out the evolutionary tree long before genome analysis was practicable. In a sense, gene arrangements are visible in the banding patterns of each chromosome. Chromosome rearrangements, especially inversions, make it possible to see which species are closely related. The results are clear. The inversions, when plotted in tree form (and independent of all other information), show a clear "flow" of species from older to newer islands. There are also cases of colonization back to older islands, and skipping of islands, but these are much less frequent. Using K-Ar dating, the present islands date from 0.4 million years ago (mya) (Mauna Kea) to 10mya (Necker). The oldest member of the Hawaiian archipelago still above the sea is Kure Atoll, which can be dated to 30 mya. The archipelago itself (produced by the Pacific plate moving over a hot spot) has existed for far longer, at least into the Cretaceous. Previous islands now beneath the sea (guyots) form the Emperor Seamount Chain. All of the native Drosophila and Scaptomyza species in Hawaii have apparently descended from a single ancestral species that colonized the islands, probably 20 million years ago. The subsequent adaptive radiation was spurred by a lack of competition and a wide variety of niches. Although it would be possible for a single gravid female to colonise an island, it is more likely to have been a group from the same species.

There are other animals and plants on the Hawaiian archipelago which have undergone similar, if less spectacular, adaptive radiations.

3.7 Depiction of karyotypes 3.7.1 Types of banding Cytogenetics employs several techniques to visualize different aspects of chromosomes:

G-banding is obtained with Giemsa stain following digestion of chromosomes with trypsin. It yields a series of lightly and darkly stained bands - the dark regions tend to be heterochromatic, late-replicating and AT rich. The light regions tend to be euchromatic, early-replicating and GC rich. This method will normally produce 300-400 bands in a normal, human genome. R-banding is the reverse of G-banding (the R stands for "reverse"). The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions).

C-banding: Giemsa binds to constitutive heterochromatin, so it stains centromeres.

Q-banding is a fluorescent pattern obtained using quinacrine for staining. The pattern of bands is very similar to that seen in G-banding.

T-banding: visualize telomeres.

Silver staining: Silver nitrate stains the nucleolar organization regionassociated protein. This yields a dark region where the silver is deposited, denoting the activity of rRNA genes within the NOR.

3.7.2 Classic karyotype cytogenetics

Karyogram from a human female lymphocyte probed for the Alu sequence using FISH. In the "classic" (depicted) karyotype, a dye, often Giemsa (G-banding), less frequently Quinacrine, is used to stain bands on the chromosomes. Giemsa is specific for the phosphate groups of DNA. Quinacrine binds to the adeninethymine-rich regions. Each chromosome has a characteristic banding pattern that helps to identify them; both chromosomes in a pair will have the same banding pattern. Karyotypes are arranged with the short arm of the chromosome on top, and the long arm on the bottom. Some karyotypes call the short and long arms p and q, respectively. In addition, the differently stained regions and sub-regions are given numerical designations from proximal to distal on the chromosome arms. For example, Cri du chat syndrome involves a deletion on the short arm of

chromosome 5. It is written as 46,XX,5p-. The critical region for this syndrome is deletion of 15.2, which is written as 46,XX,del(5)(p15.2) 3.7.3 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. Because there are a limited number of spectrally-distinct fluorophores, a combinatorial labeling method is used to generate many different colors. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope. Image processing software then assigns a pseudo color to each spectrally different combination, allowing the visualization of the individually colored chromosomes. This technique is used to identify structural chromosome aberrations in cancer cells and other disease conditions when Giemsa banding or other techniques are not accurate enough. 3.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale. Short sequences of DNA from specific loci all over the genome are isolated and enumerated. This method is also known as virtual karyotyping.

CHAPTER 4

4.1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical, as in the presence of extra or missing chromosomes, or structural, as in derivative chromosome, translocations, inversions, large-scale deletions or duplications. Numerical abnormalities, also known as aneuploidy, often occur as a result of nondisjunction during meiosis in the formation of a gamete; trisomies, in which three copies of a chromosome are present instead of the usual two, are common numerical abnormalities. Structural abnormalities often arise from errors in homologous recombination. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body, or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells. Chromosomal abnormalities that lead to disease in humans include

Turner syndrome results from a single X chromosome (45, X or 45, X0). Klinefelter syndrome, the most common male chromosomal disease, otherwise known as 47, XXY is caused by an extra X chromosome.

Edwards syndrome is caused by trisomy (three copies) of chromosome 18. Down syndrome, a common chromosomal disease, is caused by trisomy of chromosome 21.

Patau syndrome is caused by trisomy of chromosome 13. Also documented are trisomy 8, trisomy 9 and trisomy 16, although they generally do not survive to birth.

Some disorders arise from loss of just a piece of one chromosome, including

Cri du chat (cry of the cat), from a truncated short arm on chromosome 5. The name comes from the babies' distinctive cry, caused by abnormal formation of the larynx. 1p36 Deletion syndrome, from the loss of part of the short arm of chromosome 1.

Angelman syndrome 50% of cases have a segment of the long arm of chromosome 15 missing; a deletion of the maternal genes, example of imprinting disorder.

Prader-Willi syndrome 50% of cases have a segment of the long arm of chromosome 15 missing; a deletion of the paternal genes, example of imprinting disorder.

Chromosomal abnormalities can also occur in cancerous cells of an otherwise genetically normal individual; one well-documented example is the Philadelphia chromosome, a translocation mutation commonly associated with chronic myelogenous leukemia and less often with acute lymphoblastic leukemia. 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 chromosome anomaly may be detected or confirmed in this manner. Chromosome anomalies usually occur when there is an error in cell division following meiosis or mitosis. There are many types of chromosome anomalies. They can be organized into two basic groups, numerical and structural anomalies.

4.2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes), and occurs when an individual is missing either a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (Trisomy, Tetrasomy, etc.). In humans an example of a condition caused by a numerical anomaly is Down Syndrome, also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21, rather than two). Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome, an X. 4.3 Structural abnormalities When the chromosome's structure is altered. This can take several forms:

Deletions: A portion of the chromosome is missing or deleted. Known disorders in humans include Wolf-Hirschhorn syndrome, which is caused by partial deletion of the short arm of chromosome 4; and Jacobsen syndrome, also called the terminal 11q deletion disorder. Duplications: A portion of the chromosome is duplicated, resulting in extra genetic material. 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.

Translocations: When a portion of one chromosome is transferred to another chromosome. There are two main types of translocations. In a reciprocal translocation, segments from two different chromosomes have been exchanged. In a Robertsonian translocation, an entire chromosome has

attached to another at the Centromere - in humans these only occur with chromosomes 13, 14, 15, 21 and 22.

Inversions: A portion of the chromosome has broken off, turned upside down and reattached, therefore the genetic material is inverted.

Rings: A portion of a chromosome has broken off and formed a circle or ring. This can happen with or without loss of genetic material.

Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere. Chromosome instability syndromes are a group of disorders characterized by

chromosomal instability and breakage. They often lead to an increased tendency to develop certain types of malignancies. 4.3 Inheritance Most chromosome abnormalities occur as an accident in the egg or sperm, and are therefore initially not inherited. Therefore, the anomaly is present in every cell of the body. Some anomalies, however, can happen after conception, resulting in Mosaicism (where some cells have the anomaly and some do not). Chromosome anomalies can be inherited from a parent or be "de novo". This is why chromosome studies are often performed on parents when a child is found to have an anomaly. 4.4 Cytogenetics Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell, especially the chromosomes. It includes routine analysis of G-Banded chromosomes, other cytogenetic banding techniques, as well

as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). 4.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Ngeli in 1842. Their behavior in animal (salamander) cells was described by Walther Flemming, the discoverer of mitosis, in 1882. The name was coined by another German anatomist, von Waldeyer in 1888. The next stage took place after the development of genetics in the early 20th century, when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes, in contrast to their genic contents. 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, Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia, concluding an XX/XO sex determination mechanism. Painter in 1922 was not certain whether the diploid number of man was 46 or 48, at first favoring 46. He revised his opinion later from 46 to 48, and he correctly insisted on man having an XX/XY system. Considering their techniques, these results were quite remarkable. New techniques were needed to definitively solve the problem: 1. Using cells in culture
2.

Pre-treating cells in a hypotonic solution, which swells them and spreads the chromosomes

3.

Arresting mitosis in metaphase by a solution of colchicine

4. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes. Rather interestingly, the great apes have 48 chromosomes. Human chromosome 2 was formed by a merger of ancestral chromosomes, reducing the number. 4.6 Applications in biology 4.6.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. In 1931, McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes). McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize. During her cytogenetic work, McClintock discovered transposons, a find which eventually led to her Nobel Prize in 1983. 4.6.2 Natural populations of Drosophila In the 1930s, Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. persimilis from wild populations in California and neighboring states. Using Painter's technique they studied the polytene

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

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

FIG Advent of banding techniques

In the late 1960s, Caspersson developed banding techniques which differentially stain chromosomes. This allows chromosomes of otherwise equal size to be differentiated as well as to elucidate the breakpoints and constituent chromosomes involved in chromosome translocations. Deletions within one chromosome could also now be more specifically named and understood. Deletion syndromes such as DiGeorge syndrome, Prader-Willi syndrome and others were discovered to be caused by deletions in chromosome material. Diagrams identifying the chromosomes based on the banding patterns are known as cytogenetic maps. 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. 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. 4.8 Beginnings of molecular cytogenetics

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

CHAPTER 5 Techniques 5.1 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. For congenital problems usually 20 metaphase cells are scored. Future of cytogenetics Advances now focus on molecular cytogenetics including automated systems for counting the results of standard FISH preparations and techniques for virtual karyotyping, such as comparative genomic hybridization arrays, CGH and Single nucleotide polymorphism-arrays.

CHAPTER 6
MATLAB MATLAB is a high-level technical computing language and interactive environment for algorithm development, data visualization, data analysis, and numerical computation. Using MATLAB, you can solve technical computing problems faster than with traditional programming languages, such as C, C++, and FORTRAN. You can use MATLAB in a wide range of applications, including signal and image processing, communications, control design, test and measurement, financial modeling and analysis, and computational biology. Add-on toolboxes (collections of special-purpose MATLAB functions) extend the MATLAB environment to solve particular classes of problems in these application areas.

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

6.1 Image Processing The image processing step aims at image contrast enhancement and compensation of geometric distortions observed in each chromosome not related with its intrinsic shape or size. The image brightness and contrast depend on the specific tuning of the microscope and the particular geometric shape of each chromosome depends on the specific metaphase plaque from which the chromosomes were extracted. These effects must be compensated to improve the results of the pairing algorithm. The image processing step is composed of the following operations.

1) Chromosome extractionEach chromosome is isolated from the unordered karyogram. 2) Geometrical compensationThe geometric compensation, performed by using the algorithm is needed to obtain chromosomes with vertical medial axis, 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 normalizationThe features used in the comparison of chromosomes are grouped into two classes: 1) geometric based 2) pattern based (G-banding). To compare chromosomes from a band pattern point of view, geometrical and dimensional differences must be removed, or at least attenuated. Therefore, 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. 4) Intensity compensationThe metaphase plaque from which the chromosomes are extracted does not present a uniform brightness and contrast. To compensate for this inhomogeneity, the spatially scaled images are histogram equalized.

6.2 Concepts used in this phase

1) Image conversion 2) Denoising

3) Edge detection
4)

Two dimensional convolutions.

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

6.2.4 Denoising

We may define noise to be any degradation in the image signal, caused by external disturbance. If an image is being sent electronically from one place to another, via satellite or wireless transmission, or through networked cable, we may expect errors to occur in the image signal. These errors will appear on the image output in different ways depending on the type of disturbance in the signal. Usually we know what type of errors to expect, and hence the type of noise on the image; hence we can choose the most appropriate method for reducing the effects. Cleaning an image corrupted by noise is thus an important area of image restoration.

6.2.5 Edge detection Edges contain some of the most useful information in an image. We may use edges to measure the size of objects in an image; to isolate particular objects from their background; to recognize or classify objects. There is a large number of edge finding algorithms in existence, and we shall look at some of the more straightforward of them. The general Matlab command for finding edges is edge(image,'method',parameters. . . ) Where the parameters available depend on the method used 6.3 Two dimensional convolutions C = conv2(A,B) computes the two-dimensional convolution of matrices
A

and B. If one of these matrices describes a two-dimensional finite impulse response

(FIR) filter, the other matrix is filtered in two dimensions. The size of matrices, minus one. That is, if the size of then the size of C is [ma+mb-1,na+nb-1]. The indices of the center element of B are defined as floor(([mb
C = conv2(hcol,hrow,A)

in each

dimension is equal to the sum of the corresponding dimensions of the input

A

is [ma,na] and the size of

is [mb,nb],

nb]+1)/2).

convolves

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, this case is the same as
C = conv2(hcol*hrow,A). C = conv2(...,'shape')

subsection of the two-dimensional convolution, 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.bmp'); rgb2gray im2bw(im,0.7); imedfilt2(im1,[3 3]); edge(im1,'sobel');

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

end end if flag~=1 L_number(Index)=L(i,j); Index=Index+1; end flag=0; end; end end L_number; Test_number=[3,4,6,7,8,9,10,11,14,15,19,20,21,22,24,26,27,28,29,30,31,32,33,35, 36,38,39,40,41,42,43,45,48,49,50,51,52,54,55,56,57,59,60,62,65,66]; for x=1:46 [r,c] = find(L==L_number((Test_number(x)))); rc = [r c]; [sx sy]=size(rc); n1=zeros(imx,imy); for i=1:sx x1=rc(i,1); y1=rc(i,2); n1(x1,y1)=255; end %h=figure;imshow(n1,[]);

end Circumference=zeros(46,1); Arm_length=zeros(46,1); Area=zeros(46,1); for i=1:46 f=imread(strcat(num2str(i),'.bmp')); BW=im2bw(f); BW=double(BW); BW1=edge(BW,'canny'); [m n]=size(BW1); Circumference_sum=0; for x=1:m for y=1:n if BW1(x,y)==1 Circumference_sum=Circumference_sum+1; end end end Circumference(i)=Circumference_sum; f=imcomplement(f); skel=im2double(f); skel=im2bw(skel,1.5*graythresh(skel)); s=bwmorph(skel,'skel',Inf); s1=bwmorph(s,'spur',8); Arm_length_sum=0;

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

Area; Pair=zeros(46,2); for i=1:45 min=abs(Circumference(i)-Circumference(i+1))+abs(Arm_length(i)Arm_length(i+1))+abs(Area(i)-Area(i+1)); Pair(i,1)=i; Pair(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)); Pair(i,1)=i; Pair(i,2)=j; end end end for i=1:45 if Pair(i,2)==46 Pair(46,1)=46; Pair(46,2)=i; end end Pair;

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

end

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

characterizing the size, shape and band pattern; 3) training of a classifier (performed once) where similarity among chromosomes are characterized; and finally, 4) pairing. In the image processing step, the romosome images, extracted from the unordered karyogram, are processed in order to compensate for geometrical and intensity distortions, and to normalize their dimensions. This normalization is needed to make it possible the band pattern comparison between chromosomes. The features extracted from the processed images discriminate each pair with respect to their size, shape, and band pattern. Here, a novel metric distance is proposed to be used in the pairing procedure that linearly combines the distances associated with each feature. The coefficients of the linear combination are obtained through a training step using chromosomes paired manually by experts. 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. The training process consists in the estimation of each vector of coefficient , from the chromosomes in the training set, 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 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. Tests using 19 karyograms based on bone marrow cells,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, achieves a 70.10% mean classification rate. 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

performance of the classifier. Executing the algorithm on a higher quality dataset, a 76.10% classification ratewas obtained. Using 27 karyograms andworking with a limited number of classes ( 8), amean classification rate larger than 93% was obtained in all experiments. 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 addition, a new chromosome dataset with 9200 chromosomes from bone marrow cells, called LK1 , was built to provide a ground truth to test classification and pairing algorithms for this type of low image quality chromosomes. This dataset was made publicly available [29]. The results presented in this paper are promising. In fact, despite the low quality of this type of chromosomes, it was shown that it is possible to achieve comparable classification rates to the ones obtained with the classical chromosome dataset, such as Edinburgh, Copenhagen, or Philadelphia, whose images are of significantly higher quality, presenting a uniform level of condensation, and from which it is possible to extract additional features, e.g., centromere position.

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