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

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

are also found in mitochondria and chloroplasts. 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).g.+21. An example of aneuploidy is trisomy 21. The individual would have Down Syndrome and his/her karyotype would be written 47. the individual carrying the mutation is said to be aneuploid. reflecting their bacterial origins. rather than 2. in which an individual has 3. a extra copy of human chromosome 21). 1. XX (female) or 46 XY (male). Chromosomal Mutations are substantial changes in chromosome structure that are large enough to be visible by karyotyping (see lab manual) and thus typically affect more than one gene. Such individuals are called euploid and have the wild-type chromosome complement for the species. The simplest gonophores are found in viruses: these DNA or RNA molecules are short linear or circular gonophores that often lack structural proteins.XY or 47.XX. If the mutation involves only one or a few chromosomes in the genome (e. Euploid human karyotypes are 46. copies of chromosome 21.3 MUTATIONS IN CHROMOSOME NUMBER Normally. .+21.

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

[1] Bone marrow is also a key component of the lymphatic system. cytoplasm. bone marrow constitutes 4% of the total body mass of humans.1 MITOSIS Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets. bone marrow accounts for approximately 2.5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones. in two separate nuclei. Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle—the division of the mother cell into two daughter cells. The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day. It is generally followed immediately by cytokinesis. in adults weighing 65 kg (143 lbs).6 kg (5. bone marrow in large bones produces new blood cells. which divides the nuclei. which use the bone marrow vasculature as a conduit to the body's systemic circulation. producing the lymphocytes that support the body's immune system CHAPTER 2 2. . On average.1. In humans. organelles and cell membrane into two cells containing roughly equal shares of these cellular components.7 lbs).

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

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

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

chromosomes are replicated only during the S phase.1 Preprophase In plant cells only. All these phases in the interphase are highly regulated. This is achieved through the formation of a phragmosome. However. During all three phases.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. the cell grows by producing proteins and cytoplasmic organelles. the nucleus has to migrate into the center of the cell before mitosis can begin. 2. grows more and prepares for mitosis (G 2). It alternates with the much longer interphase. a transverse sheet of cytoplasm that bisects the cell along the future plane of cell . Interphase is divided into three phases: G1 (first gap). prophase is preceded by a pre-prophase stage.2.2 Phases of cell cycle and mitosis Interphase Fig 4 The cell cycle The mitotic phase is a relatively short period of the cell cycle. where the cell prepares itself for cell division. and G2 (second gap). mainly via proteins. and finally it divides (M) before restarting the cycle. Thus. continues to grow as it duplicates its chromosomes (S). a cell grows (G1). In highly vacuolated plant cells. S (synthesis).

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

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

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

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

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

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

skin and digestive tract. cells are constantly sloughed off and replaced by new ones. The phragmoplast is a microtubule structure typical for higher plants. e.1Significance Mitosis is important for the maintenance of the chromosomal set. Following are the occasions in the lives of organism where mitosis happens: 2. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell. 2.e. separating the two nuclei. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall.. Similarly.5. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis.g.5. New cells are formed by mitosis and so are exact copies of the cells being replaced. 2. zygote and also the basis of the growth of a multicellular body. This is the basis of the development of a multicellular body from a single cell i. 2. The end of cytokinesis marks the end of the M-phase.4 Regeneration .division is also driven by vesicles derived from the Golgi apparatus. whereas some green algae use a phycoplast microtubule array during cytokinesis.5.3 Cell replacement In some parts of body. which move along microtubules to the middle of the cell.2 Development and growth The number of cells within an organism increases by mitosis.5. Each daughter cell has a complete copy of the genome of its parent cell.

especially during early cellular divisions in the zygote. In non-disjunction. the process may go wrong. resulting in binucleated cells.5.7 Consequences of errors Although errors in mitosis are rare. 2. the hydra reproduces asexually by budding. Occasionally when cells experience nondisjunction. they fail to complete cell division and retain both nuclei in one cell. a condition often associated with cancer. a condition known as trisomy. . One daughter cell will receive both sister chromosomes and the other will receive none. and the latter cell having only one chromosome (the homologous chromosome).Some organisms can regenerate their parts of bodies. Mitosis continues in the cells of bud and it grows into a new individual. a chromosome may fail to separate during anaphase. The same division happens during asexual reproduction or vegetative propagation in plants. a condition known as monosomy. Mitotic errors can be especially dangerous to the organism because future offspring from this parent cell will carry the same disorder.5. sea star regenerates its lost arm through mitosis. The cells at the surface of hydra undergo mitosis and form a mass called bud. For example. 2. The production of new cells is achieved by mitosis.6 Asexual reproduction Some organisms produce genetically similar offspring through asexual reproduction. This results in the former cell having three chromosomes containing the same genes (two sisters and a homologue). These cells are considered aneuploid. For example.

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

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

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

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

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

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

2 Observations on karyotypes 3. Arresting mitosis in metaphase by a solution of colchicines 4. Usually. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. Human chromosome 2 was formed by a merger of ancestral chromosomes. 3. For humans. It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes. white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture. The sex of an unborn fetus can be determined by observation of interphase cells. Squashing the preparation on the slide forcing the chromosomes into a single plane 5.1 Staining The study of karyotypes is made possible by staining.3.2 Observations Six different characteristics of karyotypes are usually observed and compared: . such as Giemsa. Rather interestingly. a suitable dye.2.2. [16] Sometimes observations may be made on non-dividing (interphase) cells. reducing the number. the great apes have 48 chromosomes. is applied after cells have been arrested during cell division by a solution of colchicine. 3.

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

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

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

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

horsetails and psilotales) is also common. but it has been significant in some groups. and the other haploid. about 70%.4 Ploidy Ploidy is the number of complete sets of chromosomes in a cell. Polyploidy in lower plants (ferns. where there are more than two sets of homologous chromosomes in the cells. but in grasses the average is much higher. Polyploidy in animals is much less common. 21 and 22). 14. occurs mainly in plants. 15. though in this case they would not be regarded as normal members of the population. the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present. Polyploidy. FN. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%. where one sex is diploid. and in some other groups. and aneuploids are another example. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants.3 Fundamental number The fundamental number. FN ≤ 2n. Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid. 3.Endopolyploidy occurs when in adult differentiated . of a karyotype is the number of visible major chromosomal arms per set of chromosomes. Humans have FN = 82.3. 3. It is a common arrangement in the Hymenoptera.means that chromosome number can vary even within one interbreeding population. Thus. due to the presence of five acrocentric chromosome pairs (13. Haplo-diploidy. It has been of major significance in plant evolution according to Stebbins.

but the nuclei contain more than the original somatic number of chromosomes. The phenomenon occurs sporadically throughout the eukaryote kingdom from protozoa to man. 3. 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.tissues the cells have ceased to divide by mitosis. Down syndrome and Turner syndrome are examples of this. the daughter chromosomes separating from each other inside an intact nuclear membrane. This process (especially studied in insects and some higher plants such as maize) may be a developmental strategy for increasing the productivity of tissues which are highly active in biosynthesis. The cells do not always contain exact multiples (powers of two). In the endocycle (endomitosis or endoreduplication) chromosomes in a 'resting' nucleus undergo reduplication. and serves differentiation and morphogenesis in many ways. endopolyploid nuclei contain tens of thousands of chromosomes (which cannot be exactly counted). . In many instances. it is diverse and complex. See palaeopolyploidy for the investigation of ancient karyotype duplications. Abnormalities in chromosome number usually cause a defect in development. 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 chromosome number is variable from one individual to another. 3. some mantids of the genus Ameles. 4. the European shrew Sorex araneus. There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. When this happens. [41] Closer to home. and 7.000 km2). 6. reducing the number. Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups. 5. and Crocus. where the gametic (= haploid) numbers form the series x = 3. 3.Aneuploidy may also occur within a group of closely related species. Well-researched examples are the ladybird beetle Chilocorus stigma. Human chromosome 2 was formed by a merger of ancestral chromosomes. where every number from x = 3 to x = 15 is represented by at least one species. Classic examples in plants are the genus Crepis. living from rainforests to .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. the great apes have 24x2 chromosomes whereas humans have 23x2.500 sq mi (17. In about 6. that the two chromosome morphs are adapted to different habitats. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world.

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

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

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

This method is also known as virtual karyotyping.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.2) 3.XX. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope. which is written as 46. It is written as 46.XX. The critical region for this syndrome is deletion of 15.del(5)(p15.5p-.2. Image processing software then assigns a pseudo color to each spectrally different combination. allowing the visualization of the individually colored chromosomes.chromosome 5. . 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. 3. Because there are a limited number of spectrally-distinct fluorophores.7. a combinatorial labeling method is used to generate many different colors.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale.

CHAPTER 4 .

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

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

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

Therefore. Chromosome instability syndromes are a group of disorders characterized by chromosomal instability and breakage.attached to another at the Centromere . therefore the genetic material is inverted. especially the chromosomes. however. 4.in humans these only occur with chromosomes 13. 21 and 22. turned upside down and reattached. This is why chromosome studies are often performed on parents when a child is found to have an anomaly. 4. 15. • Rings: A portion of a chromosome has broken off and formed a circle or ring. Some anomalies. resulting in Mosaicism (where some cells have the anomaly and some do not). 14.3 Inheritance Most chromosome abnormalities occur as an accident in the egg or sperm.4 Cytogenetics Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell. can happen after conception. They often lead to an increased tendency to develop certain types of malignancies. and are therefore initially not inherited. • Inversions: A portion of the chromosome has broken off. This can happen with or without loss of genetic material. as well . Chromosome anomalies can be inherited from a parent or be "de novo". the anomaly is present in every cell of the body. other cytogenetic banding techniques. It includes routine analysis of G-Banded chromosomes. • Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere.

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

6. 4. Using Painter's technique they studied the polytene .1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. persimilis from wild populations in California and neighboring states. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize. the great apes have 48 chromosomes. 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). reducing the number.2 Natural populations of Drosophila In the 1930s.6 Applications in biology 4. Rather interestingly. a find which eventually led to her Nobel Prize in 1983. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. Arresting mitosis in metaphase by a solution of colchicine 4. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. McClintock discovered transposons. Human chromosome 2 was formed by a merger of ancestral chromosomes.6. During her cytogenetic work. 4.3. In 1931.

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

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

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

While radioisotope-labeled probes had been hybridized with DNA since 1969.1 Karyotyping . This change significantly increased the usage of probing techniques as fluorescent labeled probes are safer and can be used almost indefinitely.In the 1980s. 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. advances were made in molecular cytogenetics. CHAPTER 5 Techniques 5. movement was now made in using fluorescent labeled probes. Hybridizing them to chromosomal preparations using existing techniques came to be known as fluorescent in situ hybridization (FISH).

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,

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

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

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

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

. Usually we know what type of errors to expect.5 Edge detection Edges contain some of the most useful information in an image. and hence the type of noise on the image. The general Matlab command for finding edges is edge(image.'method'.B) computes the two-dimensional convolution of matrices A and B. ) Where the parameters available depend on the method used 6. If one of these matrices describes a two-dimensional finite impulse response .2. . There is a large number of edge finding algorithms in existence. Cleaning an image corrupted by noise is thus an important area of image restoration. and we shall look at some of the more straightforward of them. 6. hence we can choose the most appropriate method for reducing the effects. We may use edges to measure the size of objects in an image.2. to isolate particular objects from their background. via satellite or wireless transmission.3 Two dimensional convolutions C = conv2(A.4 Denoising We may define noise to be any degradation in the image signal.6. 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.parameters. caused by external disturbance. or through networked cable. we may expect errors to occur in the image signal. to recognize or classify objects.

If hcol is a column vector and hrow is a row vector.'sobel').(FIR) filter. The indices of the center element of B are defined as floor(([mb C = conv2(hcol..A).0. imedfilt2(im1.nb].A) C in each dimension is equal to the sum of the corresponding dimensions of the input A is [ma.hrow. minus one. The size of matrices. convolves A first with the vector hcol along the rows and then returns a with the vector hrow along the columns. edge(im1. .na+nb-1]. if the size of then the size of C is [ma+mb-1. rgb2gray im2bw(im. nb]+1)/2). C = conv2(.na] and the size of B is [mb. 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('12345.7).[3 3])..bmp'). this case is the same as C = conv2(hcol*hrow..'shape') subsection of the two-dimensional convolution. the other matrix is filtered in two dimensions. That is.

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

20.19.2).35.[]).33. for i=1:sx x1=rc(i.imshow(n1. .4. Index=Index+1.41.38. for x=1:46 [r.c] = find(L==L_number((Test_number(x)))).62.11. n1(x1.imy).39.54.end end if flag~=1 L_number(Index)=L(i.27.60.51.y1)=255.30.49. rc = [r c].7. end %h=figure.52.29.42. end.50.22.9.43.j).8.57.6.28.59. end end L_number. 36. y1=rc(i.26.10.15.21.65.1).66].56.14.32. [sx sy]=size(rc). Test_number=[3. n1=zeros(imx.31.45.55.24. end flag=0.48.40.

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

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

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

end end if flag~=1 if figure_flag~=47 subplot(23. end f1=imread(strcat(num2str(Pair(i. figure_flag=1.2. figure_flag=figure_flag+1. end f2=imread(strcat(num2str(Pair(i.2). for i=1:46 for j=1:46 if Pair(i. imshow(f2). end flag=0.figure_flag).'.1)).2.'.figure_flag).1). flag=0. if figure_flag~=47 subplot(23.1)==delete(j) flag=1.bmp')). figure_flag=figure_flag+1.delete=zeros(46. imshow(f1).bmp')). delete(figure_flag)=Pair(i.2)). .

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

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

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

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