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

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

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

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

7 lbs). [1] Bone marrow is also a key component of the lymphatic system. in two separate nuclei. bone marrow accounts for approximately 2. cytoplasm. organelles and cell membrane into two cells containing roughly equal shares of these cellular components. In humans. bone marrow constitutes 4% of the total body mass of humans. in adults weighing 65 kg (143 lbs). .1 MITOSIS Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets.6 kg (5.1. It is generally followed immediately by cytokinesis. which divides the nuclei. The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day. Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle—the division of the mother cell into two daughter cells. producing the lymphocytes that support the body's immune system CHAPTER 2 2.5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones. which use the bone marrow vasculature as a conduit to the body's systemic circulation. On average. bone marrow in large bones produces new blood cells.

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

the parent cell must make a copy of each chromosome before mitosis.Fig 3 Mitosis cell division The primary result of mitosis is the transferring of the parent cell's genome into two daughter cells. The sister chromatids are held together by a specialized region of the chromosome known as the centromere. . These two cells are identical and do not differ in any way from the original parent cell. Each chromosome now has an identical copy of itself. This occurs during the S phase of interphase. 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. 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.

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

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

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

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

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

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

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

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

The end of cytokinesis marks the end of the M-phase.e. 2.. The phragmoplast is a microtubule structure typical for higher plants.5. whereas some green algae use a phycoplast microtubule array during cytokinesis. Similarly.division is also driven by vesicles derived from the Golgi apparatus. 2. Each daughter cell has a complete copy of the genome of its parent cell. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell.3 Cell replacement In some parts of body. e. separating the two nuclei. New cells are formed by mitosis and so are exact copies of the cells being replaced.2 Development and growth The number of cells within an organism increases by mitosis.5. cells are constantly sloughed off and replaced by new ones.5. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall. skin and digestive tract.4 Regeneration . This is the basis of the development of a multicellular body from a single cell i. 2. Following are the occasions in the lives of organism where mitosis happens: 2.1Significance Mitosis is important for the maintenance of the chromosomal set. zygote and also the basis of the growth of a multicellular body.5.g. which move along microtubules to the middle of the cell.

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

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

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

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

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

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

and he correctly insisted on humans having an XX/XY system. Pretreating cells in a hypotonic solution. He revised his opinion later from 46 to 48. The name was coined by another German anatomist. New techniques were needed to definitively solve the problem: 1. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. in 1882. which swells them and spreads the chromosomes . von Waldeyer in 1888. Using cells in culture 2. in contrast to their genic contents. concluding an XX/XO sex determination mechanism. Investigation into the human karyotype took many years to settle the most basic question: how many chromosomes does a normal diploid human cell contain? In 1912.1 History of karyotype studies Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. at first favoring 46.3. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. The next stage took place after the development of genetics in the early 20th century. the discoverer of mitosis. The subsequent history of the concept can be followed in the works of Darlington and White. Painter in 1922 was not certain whether the diploid number of humans was 46 or 48. Considering their techniques. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. Their behavior in animal (salamander) cells was described by Walther Flemming. these results were quite remarkable.

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

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

and in . Between the sexes 2. The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes.Variation is often found: 1. Fig Diversity and evolution of karyotype Although the replication and transcription of DNA is highly standardized in eukaryotes. XX. XY. Between members of a population (chromosome polymorphism) 4. which are highly variable. the same cannot be said for their karyotypes. 3. Normal karyotypes for females contain two X chromosomes and are denoted 46. males have both an X and a Y chromosome denoted 46.3 The human karyotype Most (but not all) species have a standard karyotype. Mosaics or otherwise abnormal individuals. Between the germ-line and soma (between gametes and the rest of the body) 3. Any variation from the standard karyotype may lead to developmental abnormalities. Geographical variation between races 5. There is variation between species in chromosome number.

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

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

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

tissues the cells have ceased to divide by mitosis. The phenomenon occurs sporadically throughout the eukaryote kingdom from protozoa to man. 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. 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. it is diverse and complex. Down syndrome and Turner syndrome are examples of this. In many instances. which is why the simple definition 'an increase in the number of chromosome sets caused by replication without cell division' is not quite accurate. Abnormalities in chromosome number usually cause a defect in development. and serves differentiation and morphogenesis in many ways. endopolyploid nuclei contain tens of thousands of chromosomes (which cannot be exactly counted). 3.5 Aneuploidy Aneuploidy is the condition in which the chromosome number in the cells is not the typical number for the species. . See palaeopolyploidy for the investigation of ancient karyotype duplications. The cells do not always contain exact multiples (powers of two). This would give rise to a chromosome abnormality such as an extra chromosome or one or more chromosomes lost.

5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations.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. Classic examples in plants are the genus Crepis. the European shrew Sorex araneus. some mantids of the genus Ameles. In about 6. and 7. 6.000 km2). When this happens. living from rainforests to . where every number from x = 3 to x = 15 is represented by at least one species. 3. 5. Well-researched examples are the ladybird beetle Chilocorus stigma. [41] Closer to home. reducing the number. and Crocus.Aneuploidy may also occur within a group of closely related species. the chromosome number is variable from one individual to another.500 sq mi (17. the great apes have 24x2 chromosomes whereas humans have 23x2. Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups. There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world. 3. that the two chromosome morphs are adapted to different habitats. where the gametic (= haploid) numbers form the series x = 3. 4. Human chromosome 2 was formed by a merger of ancestral chromosomes.

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

The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions). The pattern of bands is very similar to that seen in G-banding. . R-banding is the reverse of G-banding (the R stands for "reverse").There are other animals and plants on the Hawaiian archipelago which have undergone similar.the dark regions tend to be heterochromatic.7. early-replicating and GC rich. • Q-banding is a fluorescent pattern obtained using quinacrine for staining. This method will normally produce 300-400 bands in a normal. if less spectacular.1 Types of banding Cytogenetics employs several techniques to visualize different aspects of chromosomes: • G-banding is obtained with Giemsa stain following digestion of chromosomes with trypsin. The light regions tend to be euchromatic. • T-banding: visualize telomeres. • • C-banding: Giemsa binds to constitutive heterochromatin. human genome.7 Depiction of karyotypes 3. 3. so it stains centromeres. It yields a series of lightly and darkly stained bands . late-replicating and AT rich. adaptive radiations.

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

The critical region for this syndrome is deletion of 15. .8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale.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.7. This technique is used to identify structural chromosome aberrations in cancer cells and other disease conditions when Giemsa banding or other techniques are not accurate enough. Short sequences of DNA from specific loci all over the genome are isolated and enumerated.2.chromosome 5. allowing the visualization of the individually colored chromosomes.del(5)(p15. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope.XX. Image processing software then assigns a pseudo color to each spectrally different combination. Because there are a limited number of spectrally-distinct fluorophores. a combinatorial labeling method is used to generate many different colors. This method is also known as virtual karyotyping.XX. 3.5p-.2) 3. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores. which is written as 46. It is written as 46.

CHAPTER 4 .

X0). trisomy 9 and trisomy 16.1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical. is caused by trisomy of chromosome 21. X or 45. Structural abnormalities often arise from errors in homologous recombination. inversions. the most common male chromosomal disease. otherwise known as 47. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body. Also documented are trisomy 8. • • Edwards syndrome is caused by trisomy (three copies) of chromosome 18. trisomies. often occur as a result of nondisjunction during meiosis in the formation of a gamete. as in the presence of extra or missing chromosomes. also known as aneuploidy.4. are common numerical abnormalities. translocations. Some disorders arise from loss of just a piece of one chromosome. or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells. although they generally do not survive to birth. Klinefelter syndrome. or structural. a common chromosomal disease. • • Patau syndrome is caused by trisomy of chromosome 13. Chromosomal abnormalities that lead to disease in humans include • • Turner syndrome results from a single X chromosome (45. in which three copies of a chromosome are present instead of the usual two. large-scale deletions or duplications. Numerical abnormalities. Down syndrome. including . XXY is caused by an extra X chromosome. as in derivative chromosome.

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

and Jacobsen syndrome. resulting in extra genetic material. which is caused by partial deletion of the short arm of chromosome 4. Known disorders in humans include Wolf-Hirschhorn syndrome. rather than two). Known human disorders include Charcot-Marie-Tooth disease type 1A which may be caused by duplication of the gene encoding peripheral myelin protein 22 (PMP22) on chromosome 17. etc. In humans an example of a condition caused by a numerical anomaly is Down Syndrome.3 Structural abnormalities When the chromosome's structure is altered. Duplications: A portion of the chromosome is duplicated. an entire chromosome has . segments from two different chromosomes have been exchanged.2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes). • • Translocations: When a portion of one chromosome is transferred to another chromosome. There are two main types of translocations. Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome. Tetrasomy. In a reciprocal translocation. This can take several forms: • Deletions: A portion of the chromosome is missing or deleted. and occurs when an individual is missing either a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (Trisomy. In a Robertsonian translocation. 4. also called the terminal 11q deletion disorder. an X.). also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21.4.

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

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

2 Natural populations of Drosophila In the 1930s. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. Arresting mitosis in metaphase by a solution of colchicine 4. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. 4. a find which eventually led to her Nobel Prize in 1983.6. the great apes have 48 chromosomes.6 Applications in biology 4. Rather interestingly. Human chromosome 2 was formed by a merger of ancestral chromosomes.3. In 1931. 4. reducing the number. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes. During her cytogenetic work. McClintock discovered transposons. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes).6. Using Painter's technique they studied the polytene .1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. persimilis from wild populations in California and neighboring states.

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

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

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

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

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

MATLAB eliminates the need for ‘for’ loops. such as declaring variables. It enables fast development and execution.1 Image Processing The image processing step aims at image contrast enhancement and compensation of geometric distortions observed in each chromosome not related with its intrinsic shape or size. and allocating memory. As a result. specifying data types. 6. . The image processing step is composed of the following operations. You can integrate your MATLAB code with other languages and applications. 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. you can program and develop algorithms faster than with traditional languages because you do not need to perform lowlevel administrative tasks.MATLAB provides a number of features for documenting and sharing your work. With the MATLAB language. 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. These effects must be compensated to improve the results of the pairing algorithm. In many cases. and distribute your MATLAB algorithms and applications.

Therefore. 6. 4) Intensity compensation—The metaphase plaque from which the chromosomes are extracted does not present a uniform brightness and contrast.2 Concepts used in this phase 1) Image conversion 2) Denoising . a dimensional scaling is performed before the pattern features is extracted to make all the chromosome with the same size and aspect ratio by interpolating the original images. To compensate for this inhomogeneity. 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) Geometrical compensation—The geometric compensation. the spatially scaled images are histogram equalized.1) Chromosome extraction—Each chromosome is isolated from the unordered karyogram. performed by using the algorithm is needed to obtain chromosomes with vertical medial axis. To compare chromosomes from a band pattern point of view. or at least attenuated. geometrical and dimensional differences must be removed.

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

3 Two dimensional convolutions C = conv2(A. we may expect errors to occur in the image signal. and hence the type of noise on the image.B) computes the two-dimensional convolution of matrices A and B. caused by external disturbance. 6. The general Matlab command for finding edges is edge(image.parameters. or through networked cable.2.5 Edge detection Edges contain some of the most useful information in an image. ) Where the parameters available depend on the method used 6. If one of these matrices describes a two-dimensional finite impulse response . These errors will appear on the image output in different ways depending on the type of disturbance in the signal.'method'. Usually we know what type of errors to expect. Cleaning an image corrupted by noise is thus an important area of image restoration.6. . . and we shall look at some of the more straightforward of them. via satellite or wireless transmission. to recognize or classify objects. to isolate particular objects from their background. There is a large number of edge finding algorithms in existence.2. If an image is being sent electronically from one place to another. hence we can choose the most appropriate method for reducing the effects. We may use edges to measure the size of objects in an image.4 Denoising We may define noise to be any degradation in the image signal.

..bmp'). imedfilt2(im1. C = conv2(. .na+nb-1].'shape') subsection of the two-dimensional convolution.0. That is. minus one.'sobel'). nb]+1)/2). rgb2gray im2bw(im. if the size of then the size of C is [ma+mb-1.A).7).nb]..(FIR) filter. convolves A first with the vector hcol along the rows and then returns a with the vector hrow along the columns. this case is the same as C = conv2(hcol*hrow. edge(im1. as specified by the shape parameter Algorithms description 1) Read the image and convert into gray 2)Remove noise 3) Background separation 4) Edge detect 5) Separate the pairs MODULE 1 PSEUDO CODE iimread('12345. the other matrix is filtered in two dimensions. The indices of the center element of B are defined as floor(([mb C = conv2(hcol.hrow. If hcol is a column vector and hrow is a row vector.A) C in each dimension is equal to the sum of the corresponding dimensions of the input A is [ma.na] and the size of B is [mb.[3 3]). The size of matrices.

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

43.24.7. end %h=figure.39.y1)=255.14.45.j). n1(x1.51.65.33.59.20.27.21.38.52.6.60. .15.2). end.[]).50.19. n1=zeros(imx. rc = [r c].32.57.1). Test_number=[3. end flag=0.imy).8.48.9.30.10.42.54.11.26.imshow(n1. y1=rc(i. 36. [sx sy]=size(rc).40. for i=1:sx x1=rc(i.55.31.49.c] = find(L==L_number((Test_number(x)))).28.62.4.29.22. end end L_number. for x=1:46 [r.end end if flag~=1 L_number(Index)=L(i.56.41.66]. Index=Index+1.35.

1. f=imcomplement(f). s=bwmorph(skel. Area=zeros(46.5*graythresh(skel)). end end end Circumference(i)=Circumference_sum. for x=1:m for y=1:n if BW1(x.1).y)==1 Circumference_sum=Circumference_sum+1. BW1=edge(BW.'canny'). Arm_length=zeros(46. [m n]=size(BW1). s1=bwmorph(s.'.1).end Circumference=zeros(46. Arm_length_sum=0.'spur'.Inf).1). skel=im2bw(skel.'skel'. BW=im2bw(f).bmp')).8). Circumference_sum=0. for i=1:46 f=imread(strcat(num2str(i). BW=double(BW). . skel=im2double(f).

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

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

1)).2.2). imshow(f2).figure_flag). figure_flag=figure_flag+1.bmp')).bmp')). flag=0. end flag=0.'. . end end if flag~=1 if figure_flag~=47 subplot(23. end f1=imread(strcat(num2str(Pair(i. figure_flag=1. figure_flag=figure_flag+1. delete(figure_flag)=Pair(i.delete=zeros(46.2)).1)==delete(j) flag=1.1). end f2=imread(strcat(num2str(Pair(i.2. for i=1:46 for j=1:46 if Pair(i. imshow(f1).figure_flag).'. if figure_flag~=47 subplot(23.

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

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

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

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