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

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

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

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

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

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

Because each resultant daughter cell should be genetically identical to the parent cell. and together the two are called sister chromatids. This occurs during the S phase of interphase. the period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs. the parent cell must make a copy of each chromosome before mitosis. Each chromosome now has an identical copy of itself. The sister chromatids are held together by a specialized region of the chromosome known as the centromere. The genome is composed of a number of chromosomes—complexes of tightly-coiled DNA that contain genetic information vital for proper cell function. .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.

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

grows more and prepares for mitosis (G 2). The phases follow one another in strict order and there are "checkpoints" that give the cell the cues to proceed from one phase to another. a transverse sheet of cytoplasm that bisects the cell along the future plane of cell . chromosomes are replicated only during the S phase. It alternates with the much longer interphase. continues to grow as it duplicates its chromosomes (S). and G2 (second gap). This is achieved through the formation of a phragmosome. Interphase is divided into three phases: G1 (first gap).2. S (synthesis). 2. prophase is preceded by a pre-prophase stage. In highly vacuolated plant cells. and finally it divides (M) before restarting the cycle. the cell grows by producing proteins and cytoplasmic organelles. the nucleus has to migrate into the center of the cell before mitosis can begin. Thus. mainly via proteins.1 Preprophase In plant cells only. During all three phases. where the cell prepares itself for cell division.2 Phases of cell cycle and mitosis Interphase Fig 4 The cell cycle The mitotic phase is a relatively short period of the cell cycle.2. All these phases in the interphase are highly regulated. a cell grows (G1). However.

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

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

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

. Then the two centrosomes start pulling the chromosomes through their attached centromeres towards the two ends of the cell. In certain types of cells. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. The centrosome acts as the "reel" that draws in the spindle fibers or "fishing line". in some sense. Metaphase comes from the Greek meaning "after. All chromosomes (blue) but one have arrived at the metaphase plate.In the fishing pole analogy. the kinetochore would be the "hook" that catches a sister chromatid or "fish". the chromosomes come under longitudinal tension from the two ends of the cell. convene along the metaphase plate or equatorial plane. only roughly lining up along the midline." Microtubules find and attach to kinetochores in prometaphase. 2.3 Metaphase A cell in late metaphase. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. 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. The centromeres of the chromosomes. As a result. analogous to a tug-of-war between people of equal strength.

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

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

each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell. whereas some green algae use a phycoplast microtubule array during cytokinesis. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall. 2. e. 2. zygote and also the basis of the growth of a multicellular body.division is also driven by vesicles derived from the Golgi apparatus.3 Cell replacement In some parts of body.e.1Significance Mitosis is important for the maintenance of the chromosomal set.5. The end of cytokinesis marks the end of the M-phase.4 Regeneration . Similarly. Following are the occasions in the lives of organism where mitosis happens: 2.5.5. cells are constantly sloughed off and replaced by new ones.5. 2. This is the basis of the development of a multicellular body from a single cell i.. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis. New cells are formed by mitosis and so are exact copies of the cells being replaced. which move along microtubules to the middle of the cell.g. separating the two nuclei. Each daughter cell has a complete copy of the genome of its parent cell.2 Development and growth The number of cells within an organism increases by mitosis. skin and digestive tract. The phragmoplast is a microtubule structure typical for higher plants.

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

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

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

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

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

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

Considering their techniques. at first favoring 46. in 1882. The subsequent history of the concept can be followed in the works of Darlington and White. the discoverer of mitosis. concluding an XX/XO sex determination mechanism. Pretreating cells in a hypotonic solution. these results were quite remarkable. He revised his opinion later from 46 to 48. The next stage took place after the development of genetics in the early 20th century. Investigation into the human karyotype took many years to settle the most basic question: how many chromosomes does a normal diploid human cell contain? In 1912. Painter in 1922 was not certain whether the diploid number of humans was 46 or 48. New techniques were needed to definitively solve the problem: 1. The name was coined by another German anatomist. Their behavior in animal (salamander) cells was described by Walther Flemming. which swells them and spreads the chromosomes .3. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. von Waldeyer in 1888. in contrast to their genic contents. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. and he correctly insisted on humans having an XX/XY system. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes.1 History of karyotype studies Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. Using cells in culture 2.

2. is applied after cells have been arrested during cell division by a solution of colchicine.3. Rather interestingly. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. reducing the number. [16] Sometimes observations may be made on non-dividing (interphase) cells. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. Arresting mitosis in metaphase by a solution of colchicines 4.2 Observations Six different characteristics of karyotypes are usually observed and compared: . 3. a suitable dye.1 Staining The study of karyotypes is made possible by staining. such as Giemsa. It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes. The sex of an unborn fetus can be determined by observation of interphase cells.2 Observations on karyotypes 3. white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture. 3. Human chromosome 2 was formed by a merger of ancestral chromosomes. Usually.2. the great apes have 48 chromosomes. For humans.

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

Fig Diversity and evolution of karyotype Although the replication and transcription of DNA is highly standardized in eukaryotes. The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. Mosaics or otherwise abnormal individuals. Geographical variation between races 5.3 The human karyotype Most (but not all) species have a standard karyotype. which are highly variable. the same cannot be said for their karyotypes. Any variation from the standard karyotype may lead to developmental abnormalities. males have both an X and a Y chromosome denoted 46. 3. Between members of a population (chromosome polymorphism) 4. Normal karyotypes for females contain two X chromosomes and are denoted 46. Between the sexes 2. There is variation between species in chromosome number. XY. and in .Variation is often found: 1. XX. Between the germ-line and soma (between gametes and the rest of the body) 3.

entire chromosomes are eliminated during development. But. . In a review. 3. Although much is known about karyotypes at the descriptive level. Chromatin diminution (founding father: Theodor Boveri). In this process.detailed organization. "We have a very poor understanding of the causes of karyotype evolution. This process is a carefully organised genome rearrangement where new telomeres are constructed and certain heterochromatin regions are lost. which were previously inexplicable. and it is clear that changes in karyotype organization have had effects on the evolutionary course of many species. used in conjunction with other phylogenetic data. karyotypic fissioning may help to explain dramatic differences in diploid numbers between closely related species. This variation provides the basis for a range of studies in evolutionary cytology. In A. as in many sciarid flies. found in some copepods and roundworms such as Ascaris suum.3.1 Changes during development Instead of the usual gene repression. or other kinds of visible adjustment to the karyotype. some organisms go in for large-scale elimination of heterochromatin. it is quite unclear what the general significance might be. it is unlikely that one process or the other can independently account for the wide range of karyotype structures that are observed. Chromosome elimination. despite their construction from the same macromolecules. despite many careful investigations. Godfrey and Masters conclude: "In our view... portions of the chromosomes are cast away in particular cells.. the general significance of karyotype evolution is obscure. In some species. In some cases there is even significant variation within species..

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

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

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

living from rainforests to . 3. where every number from x = 3 to x = 15 is represented by at least one species.Aneuploidy may also occur within a group of closely related species.500 sq mi (17. reducing the number. and Crocus. that the two chromosome morphs are adapted to different habitats. Classic examples in plants are the genus Crepis. Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups. When this happens. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world.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. [41] Closer to home. the European shrew Sorex araneus.000 km2). some mantids of the genus Ameles. There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast. 4. and 7. 6.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. where the gametic (= haploid) numbers form the series x = 3. In about 6. 3. the chromosome number is variable from one individual to another. the great apes have 24x2 chromosomes whereas humans have 23x2. Well-researched examples are the ladybird beetle Chilocorus stigma. 5. Human chromosome 2 was formed by a merger of ancestral chromosomes.

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

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

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

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. which is written as 46. Image processing software then assigns a pseudo color to each spectrally different combination. This method is also known as virtual karyotyping. allowing the visualization of the individually colored chromosomes. The critical region for this syndrome is deletion of 15.7.2) 3. 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.XX.XX. It is written as 46.2. 3.3 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.chromosome 5. .8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale. Because there are a limited number of spectrally-distinct fluorophores.del(5)(p15.5p-. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores.

CHAPTER 4 .

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

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

an X. Tetrasomy. and occurs when an individual is missing either a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (Trisomy. rather than two). In a reciprocal translocation. 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.4. segments from two different chromosomes have been exchanged.). This can take several forms: • Deletions: A portion of the chromosome is missing or deleted.2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes). 4. which is caused by partial deletion of the short arm of chromosome 4. Duplications: A portion of the chromosome is duplicated. etc. and Jacobsen syndrome. 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. Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome. Known disorders in humans include Wolf-Hirschhorn syndrome. an entire chromosome has . • • Translocations: When a portion of one chromosome is transferred to another chromosome. also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21. In a Robertsonian translocation. also called the terminal 11q deletion disorder. There are two main types of translocations. resulting in extra genetic material.

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

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

It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. Using Painter's technique they studied the polytene . 4. the great apes have 48 chromosomes. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes). Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. In 1931. Human chromosome 2 was formed by a merger of ancestral chromosomes. Rather interestingly.3.6. a find which eventually led to her Nobel Prize in 1983. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize. reducing the number.6 Applications in biology 4. Arresting mitosis in metaphase by a solution of colchicine 4.6. persimilis from wild populations in California and neighboring states. McClintock discovered transposons.2 Natural populations of Drosophila In the 1930s. 4.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. During her cytogenetic work.

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

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

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

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

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,

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

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

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

You can perform certain conversions just using MATLAB syntax.3) Edge detection 4) Two dimensional convolutions.1 Image conversion The toolbox includes many functions that you can use to convert an image from one type to another.I. 6. The resulting true color image has identical matrices for the red. green. and blue planes. 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. . there are other functions that return a different image type as part of the operation they perform. if you want to filter a color image that is stored as an indexed image. For example. MATLAB filters the intensity values in the image. For example. MATLAB simply applies the filter to the indices in the indexed image matrix. and the results might not be meaningful.I. so the image displays as shades of gray. If you attempt to filter the indexed image. the region of interest functions returns a binary image that you can use to mask an image for filtering or for other operations. listed in the following table. For example. In addition to these image type conversion functions.I). RGB = cat (3.2. as is appropriate. you must first convert it to true color format.

6.B) computes the two-dimensional convolution of matrices A and B. and we shall look at some of the more straightforward of them. and hence the type of noise on the image. to recognize or classify objects.5 Edge detection Edges contain some of the most useful information in an image.2. to isolate particular objects from their background. via satellite or wireless transmission. . There is a large number of edge finding algorithms in existence.2.3 Two dimensional convolutions C = conv2(A. The general Matlab command for finding edges is edge(image. ) Where the parameters available depend on the method used 6. we may expect errors to occur in the image signal. or through networked cable. caused by external disturbance.parameters.'method'. hence we can choose the most appropriate method for reducing the effects.4 Denoising We may define noise to be any degradation in the image signal. Cleaning an image corrupted by noise is thus an important area of image restoration. . 6. If one of these matrices describes a two-dimensional finite impulse response . We may use edges to measure the size of objects in an image. If an image is being sent electronically from one place to another. 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.

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

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

36.62.30. rc = [r c].39.end end if flag~=1 L_number(Index)=L(i. .43.45.35.19.21.42. Index=Index+1.49.4.7.38.51.31.48.55.65.y1)=255.56.26.10. end end L_number. end %h=figure.15.52.20.8.22. end flag=0.33.66].60.14. for x=1:46 [r. y1=rc(i.[]).24.2).41.29. end.57. [sx sy]=size(rc).27.28. Test_number=[3. for i=1:sx x1=rc(i.11.50.6.imshow(n1.59.32.c] = find(L==L_number((Test_number(x)))).1).j).40. n1=zeros(imx. n1(x1.9.54.imy).

[m n]=size(BW1). BW1=edge(BW. skel=im2double(f).'spur'.Inf). for i=1:46 f=imread(strcat(num2str(i).1.8). Circumference_sum=0. .'. s=bwmorph(skel. f=imcomplement(f).5*graythresh(skel)).1).1). Arm_length=zeros(46.1). Area=zeros(46. end end end Circumference(i)=Circumference_sum.'canny').bmp')). BW=im2bw(f).end Circumference=zeros(46. for x=1:m for y=1:n if BW1(x. skel=im2bw(skel. BW=double(BW).'skel'. Arm_length_sum=0.y)==1 Circumference_sum=Circumference_sum+1. s1=bwmorph(s.

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

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

figure_flag=figure_flag+1.bmp')). figure_flag=figure_flag+1.figure_flag). flag=0. end end if flag~=1 if figure_flag~=47 subplot(23. .'.2)).delete=zeros(46. if figure_flag~=47 subplot(23.1)). imshow(f2).1)==delete(j) flag=1. end flag=0.1).2). for i=1:46 for j=1:46 if Pair(i.bmp')).figure_flag).'. delete(figure_flag)=Pair(i. imshow(f1).2. end f2=imread(strcat(num2str(Pair(i.2. figure_flag=1. end f1=imread(strcat(num2str(Pair(i.

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

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

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

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