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

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

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

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

6 kg (5. producing the lymphocytes that support the body's immune system CHAPTER 2 2. in two separate nuclei. It is generally followed immediately by cytokinesis. The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day. In humans. in adults weighing 65 kg (143 lbs).1.1 MITOSIS Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets. [1] Bone marrow is also a key component of the lymphatic system. bone marrow constitutes 4% of the total body mass of humans.7 lbs). which use the bone marrow vasculature as a conduit to the body's systemic circulation. bone marrow in large bones produces new blood cells.5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones. cytoplasm. Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle—the division of the mother cell into two daughter cells. . which divides the nuclei. On average. organelles and cell membrane into two cells containing roughly equal shares of these cellular components. bone marrow accounts for approximately 2.

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

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

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

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

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

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

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

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

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

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

4 Regeneration .5. skin and digestive tract. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell.e. The end of cytokinesis marks the end of the M-phase. cells are constantly sloughed off and replaced by new ones.2 Development and growth The number of cells within an organism increases by mitosis. Following are the occasions in the lives of organism where mitosis happens: 2. e.3 Cell replacement In some parts of body.5.division is also driven by vesicles derived from the Golgi apparatus. which move along microtubules to the middle of the cell. 2. separating the two nuclei. Each daughter cell has a complete copy of the genome of its parent cell.. New cells are formed by mitosis and so are exact copies of the cells being replaced. 2.5. whereas some green algae use a phycoplast microtubule array during cytokinesis. zygote and also the basis of the growth of a multicellular body.1Significance Mitosis is important for the maintenance of the chromosomal set. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall. 2. Similarly. The phragmoplast is a microtubule structure typical for higher plants.5. 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.g.

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

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

In certain types of cells. This process may also be referred to as endoreduplication and the cells as endoploid. only roughly lining up along the middleline. Preceded by events in prometaphase and followed by anaphase.2. 2. Early events of metaphase can .6 Endomitosis Endomitosis is a variant of mitosis without nuclear or cellular division. microtubules formed in prophase have already found and attached themselves to kinetochores in 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. analogous to a tug of war between equally strong people. carrying genetic information. from the ancient Greek(between) and (stage). align in the middle of the cell before being separated into each of the two daughter cells.7 Metaphase Metaphase. resulting in cells with many copies of the same chromosome occupying a single nucleus. Metaphase accounts for approximately 4% of the cell cycle's duration. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. An example of a cell that goes through endomitosis is the megakaryocyte. The centromeres of the chromosomes convene themselves on the metaphase plate (or equatorial plate). is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes.

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

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. which may lead to chimeric oncogenes. Malignant cells from solid tumors or leukemia samples can also be used for cytogenetic analysis to generate metaphase preparations. for example. such as bcr-abl in chronic myelogenous leukemia. .methods like FISH and as a hybridization matrix for comparative genomic hybridization (CGH) experiments.

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

He revised his opinion later from 46 to 48. Using cells in culture 2. in contrast to their genic contents. The subsequent history of the concept can be followed in the works of Darlington and White. and he correctly insisted on humans having an XX/XY system. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. the discoverer of mitosis. 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. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. which swells them and spreads the chromosomes . von Waldeyer in 1888. Pretreating cells in a hypotonic solution. Their behavior in animal (salamander) cells was described by Walther Flemming.1 History of karyotype studies Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. Painter in 1922 was not certain whether the diploid number of humans was 46 or 48. The next stage took place after the development of genetics in the early 20th century. these results were quite remarkable. at first favoring 46. concluding an XX/XO sex determination mechanism. The name was coined by another German anatomist. Considering their techniques. New techniques were needed to definitively solve the problem: 1. in 1882.3.

[16] Sometimes observations may be made on non-dividing (interphase) cells.1 Staining The study of karyotypes is made possible by staining. is applied after cells have been arrested during cell division by a solution of colchicine. a suitable dye. Rather interestingly. Squashing the preparation on the slide forcing the chromosomes into a single plane 5.3. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. The sex of an unborn fetus can be determined by observation of interphase cells. the great apes have 48 chromosomes. 3.2 Observations on karyotypes 3.2. It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes. 3. reducing the number. For humans. such as Giemsa. Human chromosome 2 was formed by a merger of ancestral chromosomes.2 Observations Six different characteristics of karyotypes are usually observed and compared: . white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture. Usually. Arresting mitosis in metaphase by a solution of colchicines 4.2.

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

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

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

Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a mere 372 chromosomes. In human females some 15% of somatic cells escape inactivation. the high record would be somewhere amongst the ferns. In placental mammals.2 Number of chromosomes in a set A spectacular example of variability between closely related species is the muntjac. where the haploid n = 1. was found to be 46. In marsupials it is always the paternal X which is inactivated. Muntiacus muntjak. When they looked at the karyotype of the closely related Indian muntjac.. But when they obtained a couple more specimens they confirmed [their findings]" Hsu p73-4 The number of chromosomes in the karyotype between (relatively) unrelated species is hugely variable.. The inactivation of one X chromosome takes place during the early development of mammals (see Barr body and dosage compensation).3. The existence of supernumerary or B chromosomes . all the somatic cell precursors undergo chromatin diminution.suum. all telocentric.. Muntiacus reevesi. which was investigated by Kurt Benirschke and his colleague Doris Wurster. male = 7 chromosomes.. the inactivation is random as between the two Xs. Xinactivation. 3. thus the mammalian female is a mosaic in respect of her X chromosomes. 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. they were astonished to find it had female = 6. with the Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes. "They simply could not believe what they saw. The diploid number of the Chinese muntjac.

but in grasses the average is much higher. of a karyotype is the number of visible major chromosomal arms per set of chromosomes. 14. Humans have FN = 82. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%.Endopolyploidy occurs when in adult differentiated . the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present. occurs mainly in plants. and the other haploid. but it has been significant in some groups. due to the presence of five acrocentric chromosome pairs (13. Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid. about 70%.3. where one sex is diploid. where there are more than two sets of homologous chromosomes in the cells. It is a common arrangement in the Hymenoptera. horsetails and psilotales) is also common. and in some other groups. 3. Thus. Polyploidy. 3. FN ≤ 2n.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. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants. Haplo-diploidy. 21 and 22). It has been of major significance in plant evolution according to Stebbins. Polyploidy in lower plants (ferns. 15. and aneuploids are another example.3 Fundamental number The fundamental number. FN.4 Ploidy Ploidy is the number of complete sets of chromosomes in a cell. Polyploidy in animals is much less common.

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. Down syndrome and Turner syndrome are examples of this. 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. endopolyploid nuclei contain tens of thousands of chromosomes (which cannot be exactly counted).tissues the cells have ceased to divide by mitosis. In many instances. and serves differentiation and morphogenesis in many ways. The phenomenon occurs sporadically throughout the eukaryote kingdom from protozoa to man. 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 daughter chromosomes separating from each other inside an intact nuclear membrane. 3. See palaeopolyploidy for the investigation of ancient karyotype duplications. Abnormalities in chromosome number usually cause a defect in development. This would give rise to a chromosome abnormality such as an extra chromosome or one or more chromosomes lost.5 Aneuploidy Aneuploidy is the condition in which the chromosome number in the cells is not the typical number for the species. . it is diverse and complex.

the European shrew Sorex araneus. and 7. the great apes have 24x2 chromosomes whereas humans have 23x2. the chromosome number is variable from one individual to another. reducing the number. 6. where the gametic (= haploid) numbers form the series x = 3. Human chromosome 2 was formed by a merger of ancestral chromosomes. and Crocus. 3. Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups. living from rainforests to . When this happens.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.Aneuploidy may also occur within a group of closely related species. There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast. some mantids of the genus Ameles. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world. that the two chromosome morphs are adapted to different habitats.500 sq mi (17. where every number from x = 3 to x = 15 is represented by at least one species. In about 6. 5.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations.000 km2). 4. [41] Closer to home. 3. Classic examples in plants are the genus Crepis. Well-researched examples are the ladybird beetle Chilocorus stigma.

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

The light regions tend to be euchromatic. The pattern of bands is very similar to that seen in G-banding. • Q-banding is a fluorescent pattern obtained using quinacrine for staining. human genome. • • C-banding: Giemsa binds to constitutive heterochromatin. 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. This method will normally produce 300-400 bands in a normal. late-replicating and AT rich. .the dark regions tend to be heterochromatic.7. The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions).7 Depiction of karyotypes 3. 3.1 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. • T-banding: visualize telomeres. so it stains centromeres. if less spectacular. adaptive radiations. early-replicating and GC rich. It yields a series of lightly and darkly stained bands .

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

. allowing the visualization of the individually colored chromosomes. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope.2. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores.7. 3.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale. 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. which is written as 46. Because there are a limited number of spectrally-distinct fluorophores. Image processing software then assigns a pseudo color to each spectrally different combination. This method is also known as virtual karyotyping.del(5)(p15.3 Spectral karyotype (SKY technique) Spectral karyotyping is a molecular cytogenetic technique used to simultaneously visualize all the pairs of chromosomes in an organism in different colors.chromosome 5. Short sequences of DNA from specific loci all over the genome are isolated and enumerated. a combinatorial labeling method is used to generate many different colors.XX.XX. It is written as 46.5p-.2) 3. The critical region for this syndrome is deletion of 15.

CHAPTER 4 .

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

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

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

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

Using cells in culture 2. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. the discoverer of mitosis. concluding an XX/XO sex determination mechanism. He revised his opinion later from 46 to 48. 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 . when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. New techniques were needed to definitively solve the problem: 1. von Waldeyer in 1888.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). these results were quite remarkable. The next stage took place after the development of genetics in the early 20th century. 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.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. at first favoring 46. Considering their techniques. 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. in 1882. 4. Pre-treating cells in a hypotonic solution. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. in contrast to their genic contents.

Cutting up a photomicrograph and arranging the result into an indisputable karyogram. a find which eventually led to her Nobel Prize in 1983. reducing the number. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize. McClintock discovered transposons.2 Natural populations of Drosophila In the 1930s. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes).3.6.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. 4. 4.6 Applications in biology 4. persimilis from wild populations in California and neighboring states.6. During her cytogenetic work. Arresting mitosis in metaphase by a solution of colchicine 4. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. Rather interestingly. In 1931. the great apes have 48 chromosomes. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. Human chromosome 2 was formed by a merger of ancestral chromosomes. Using Painter's technique they studied the polytene .

4. Stocks containing inversions at a known initial frequency can be maintained in controlled conditions. Down syndrome is also referred to as trisomy 21.7 Human abnormalities and medical applications In the event of procedures which allowed easy enumeration of chromosomes. breeding and sampling whilst preventing escape. 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. Lejeune discovered patients with Down syndrome had an extra copy of chromosome 21.chromosomes and discovered that the wild populations were polymorphic for chromosomal inversions. Dobzhansky bred populations in population cages. Evidence rapidly accumulated to show that natural selection was responsible. as with most polymorphisms. All the flies look alike whatever inversions they carry: this is an example of a cryptic polymorphism. such as Down's syndrome. It was found that the various chromosome types do not fluctuate at random. but adjust to certain frequencies at which they become stabilised. Using a method invented by L'Heretier and Teissier. In some congenital disorders. 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. as they would if selectively neutral. This had the benefit of eliminating migration as a possible explanation of the results. cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy. In 1959. . discoveries were quickly made related to aberrant chromosomes or chromosome number.

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

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

1 Karyotyping . 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. advances were made in molecular cytogenetics. CHAPTER 5 Techniques 5.In the 1980s. movement was now made in using fluorescent labeled probes. Further advances in micromanipulation and examination of chromosomes led to the technique of chromosome microdissection whereby aberrations in chromosomal structure could be isolated. cloned and studied in ever greater detail. Hybridizing them to chromosomal preparations using existing techniques came to be known as fluorescent in situ hybridization (FISH).

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,

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

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

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

you must first convert it to true color format. When you apply the filter to the true color image. For example. . MATLAB filters the intensity values in the image.2. The resulting true color image has identical matrices for the red.I. 6.3) Edge detection 4) Two dimensional convolutions. You can perform certain conversions just using MATLAB syntax. if you want to filter a color image that is stored as an 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. 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. there are other functions that return a different image type as part of the operation they perform. In addition to these image type conversion functions. so the image displays as shades of gray. For example. If you attempt to filter the indexed image. listed in the following table.1 Image conversion The toolbox includes many functions that you can use to convert an image from one type to another.I). green. MATLAB simply applies the filter to the indices in the indexed image matrix. RGB = cat (3. For example.I. and the results might not be meaningful. as is appropriate.

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. caused by external disturbance.'method'.5 Edge detection Edges contain some of the most useful information in an image.2. via satellite or wireless transmission. 6. Cleaning an image corrupted by noise is thus an important area of image restoration. The general Matlab command for finding edges is edge(image.parameters. .B) computes the two-dimensional convolution of matrices A and B. These errors will appear on the image output in different ways depending on the type of disturbance in the signal. to isolate particular objects from their background. hence we can choose the most appropriate method for reducing the effects. or through networked cable. to recognize or classify objects. and hence the type of noise on the image. .4 Denoising We may define noise to be any degradation in the image signal. and we shall look at some of the more straightforward of them. If one of these matrices describes a two-dimensional finite impulse response . ) Where the parameters available depend on the method used 6. There is a large number of edge finding algorithms in existence. we may expect errors to occur in the image signal. Usually we know what type of errors to expect.6.3 Two dimensional convolutions C = conv2(A.2.

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

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

66].60.[]).38. Index=Index+1. for i=1:sx x1=rc(i.55.65.20.1).14.c] = find(L==L_number((Test_number(x)))). rc = [r c]. for x=1:46 [r.28.26.19. end flag=0.57.43. y1=rc(i.29.48. 36.62.39.42.59. end %h=figure.33.10.y1)=255.40.end end if flag~=1 L_number(Index)=L(i.imy).56.8.32.4.9. .54.49.41. n1=zeros(imx. end. end end L_number.30.24.51.31.j).2).27.imshow(n1.45.11.50.15.22. Test_number=[3.21. n1(x1.35. [sx sy]=size(rc).7.6.52.

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

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

2)=i+1. Pair(i. . 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)==46 Pair(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)).1)=i.2)=i. Pair=zeros(46. Pair(i. Pair(46.2)=j.1)=i. end end Pair. Pair(i. end end end for i=1:45 if Pair(i.Area.1)=46.2).

bmp')). . for i=1:46 for j=1:46 if Pair(i. figure_flag=1.figure_flag). imshow(f2). end f2=imread(strcat(num2str(Pair(i.1)).bmp')).figure_flag). end f1=imread(strcat(num2str(Pair(i. figure_flag=figure_flag+1. flag=0. delete(figure_flag)=Pair(i.2).'.2)).2. figure_flag=figure_flag+1.delete=zeros(46.1)==delete(j) flag=1.'. if figure_flag~=47 subplot(23. end end if flag~=1 if figure_flag~=47 subplot(23. end flag=0.1).2. imshow(f1).

dimensions and banding profiles. based on the MI. to improve the discriminative power of the pairing algorithm with respect to the the G-banding pattern. and Philadelphia. 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. such as. The proposed algorithm is based on the traditional features extracted from the karyogram. plus a new one. 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. Copenhagen. in the scope of karyotyping process used in cytogentic analysis. 2) feature extraction from the processed images . 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.end CONCLUTION In this paper.

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

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

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