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

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.XX. Such individuals are called euploid and have the wild-type chromosome complement for the species. 1. rather than 2.g. The individual would have Down Syndrome and his/her karyotype would be written 47. copies of chromosome 21. An example of aneuploidy is trisomy 21. in which an individual has 3.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). the individual carrying the mutation is said to be aneuploid.+21. If the mutation involves only one or a few chromosomes in the genome (e. reflecting their bacterial origins. XX (female) or 46 XY (male). The simplest gonophores are found in viruses: these DNA or RNA molecules are short linear or circular gonophores that often lack structural proteins.+21. a extra copy of human chromosome 21). Euploid human karyotypes are 46.3 MUTATIONS IN CHROMOSOME NUMBER Normally.XY or 47. .

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

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

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

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

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

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

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

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

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

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

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

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

cells are constantly sloughed off and replaced by new ones. Each daughter cell has a complete copy of the genome of its parent cell. zygote and also the basis of the growth of a multicellular body.3 Cell replacement In some parts of body. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell. The end of cytokinesis marks the end of the M-phase. Following are the occasions in the lives of organism where mitosis happens: 2. 2. New cells are formed by mitosis and so are exact copies of the cells being replaced.division is also driven by vesicles derived from the Golgi apparatus.e.5.4 Regeneration . whereas some green algae use a phycoplast microtubule array during cytokinesis.2 Development and growth The number of cells within an organism increases by mitosis. 2. which move along microtubules to the middle of the cell.g.5.1Significance Mitosis is important for the maintenance of the chromosomal set. 2. e.. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis.5. separating the two nuclei. The phragmoplast is a microtubule structure typical for higher plants. This is the basis of the development of a multicellular body from a single cell i. skin and digestive tract.5. Similarly. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall.

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

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

This process may also be referred to as endoreduplication and the cells as endoploid. 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. Preceded by events in prometaphase and followed by anaphase. microtubules formed in prophase have already found and attached themselves to kinetochores in metaphase. only roughly lining up along the middleline. 2.7 Metaphase Metaphase.6 Endomitosis Endomitosis is a variant of mitosis without nuclear or cellular division. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. An example of a cell that goes through endomitosis is the megakaryocyte. carrying genetic information. resulting in cells with many copies of the same chromosome occupying a single nucleus. from the ancient Greek(between) and (stage). Early events of metaphase can . In certain types of cells.2. an imaginary line that is equidistant from the two centrosome poles. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. analogous to a tug of war between equally strong people. Metaphase accounts for approximately 4% of the cell cycle's duration. align in the middle of the cell before being separated into each of the two daughter cells.

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

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

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

Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. concluding an XX/XO sex determination mechanism. Pretreating cells in a hypotonic solution.1 History of karyotype studies Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. von Waldeyer in 1888. the discoverer of mitosis. The next stage took place after the development of genetics in the early 20th century. these results were quite remarkable. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. in 1882. Considering their techniques. which swells them and spreads the chromosomes . when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. He revised his opinion later from 46 to 48.3. in contrast to their genic contents. The name was coined by another German anatomist. Painter in 1922 was not certain whether the diploid number of humans was 46 or 48. Using cells in culture 2. The subsequent history of the concept can be followed in the works of Darlington and White. Investigation into the human karyotype took many years to settle the most basic question: how many chromosomes does a normal diploid human cell contain? In 1912. Their behavior in animal (salamander) cells was described by Walther Flemming. and he correctly insisted on humans having an XX/XY system. at first favoring 46. New techniques were needed to definitively solve the problem: 1.

[16] Sometimes observations may be made on non-dividing (interphase) cells. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. 3.2 Observations Six different characteristics of karyotypes are usually observed and compared: . Human chromosome 2 was formed by a merger of ancestral chromosomes. Arresting mitosis in metaphase by a solution of colchicines 4. The sex of an unborn fetus can be determined by observation of interphase cells. a suitable dye. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes.2. Usually. 3.3. Rather interestingly.2 Observations on karyotypes 3. For humans. white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture. is applied after cells have been arrested during cell division by a solution of colchicine.1 Staining The study of karyotypes is made possible by staining.2. such as Giemsa. the great apes have 48 chromosomes. reducing the number.

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

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

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

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

Polyploidy.4 Ploidy Ploidy is the number of complete sets of chromosomes in a cell. It is a common arrangement in the Hymenoptera. 3. Haplo-diploidy. occurs mainly in plants. of a karyotype is the number of visible major chromosomal arms per set of chromosomes. Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid. though in this case they would not be regarded as normal members of the population.3. Polyploidy in lower plants (ferns. 15. 3.3 Fundamental number The fundamental number. due to the presence of five acrocentric chromosome pairs (13. where one sex is diploid. about 70%. and aneuploids are another example. It has been of major significance in plant evolution according to Stebbins. Humans have FN = 82. where there are more than two sets of homologous chromosomes in the cells.means that chromosome number can vary even within one interbreeding population. and the other haploid. 14. Thus. Polyploidy in animals is much less common. but it has been significant in some groups.Endopolyploidy occurs when in adult differentiated . and in some other groups. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants. 21 and 22). but in grasses the average is much higher. FN ≤ 2n. the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present. horsetails and psilotales) is also common. FN. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%.

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

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

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

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

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

This method is also known as virtual karyotyping.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale. Because there are a limited number of spectrally-distinct fluorophores. Short sequences of DNA from specific loci all over the genome are isolated and enumerated. allowing the visualization of the individually colored chromosomes.2) 3.XX.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. a combinatorial labeling method is used to generate many different colors. which is written as 46. The critical region for this syndrome is deletion of 15. . Image processing software then assigns a pseudo color to each spectrally different combination. It is written as 46.7. This technique is used to identify structural chromosome aberrations in cancer cells and other disease conditions when Giemsa banding or other techniques are not accurate enough.XX.chromosome 5. 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.5p-.del(5)(p15.2. 3.

CHAPTER 4 .

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. in which three copies of a chromosome are present instead of the usual two. the most common male chromosomal disease. • • Patau syndrome is caused by trisomy of chromosome 13. Down syndrome. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body.4. as in the presence of extra or missing chromosomes. Klinefelter syndrome. although they generally do not survive to birth. a common chromosomal disease. Structural abnormalities often arise from errors in homologous recombination. X0). also known as aneuploidy. trisomy 9 and trisomy 16. often occur as a result of nondisjunction during meiosis in the formation of a gamete. is caused by trisomy of chromosome 21. or structural. including . X or 45. large-scale deletions or duplications. Also documented are trisomy 8. trisomies. otherwise known as 47. or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells. • • Edwards syndrome is caused by trisomy (three copies) of chromosome 18.1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical. as in derivative chromosome. are common numerical abnormalities. XXY is caused by an extra X chromosome. Numerical abnormalities. translocations. inversions.

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

This can take several forms: • Deletions: A portion of the chromosome is missing or deleted. Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome. In humans an example of a condition caused by a numerical anomaly is Down Syndrome. etc. an X. resulting in extra genetic material. also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21. • • Translocations: When a portion of one chromosome is transferred to another chromosome. In a Robertsonian translocation. which is caused by partial deletion of the short arm of chromosome 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. Known disorders in humans include Wolf-Hirschhorn syndrome.). and Jacobsen syndrome. Duplications: A portion of the chromosome is duplicated. There are two main types of translocations. also called the terminal 11q deletion disorder.4.2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes). 4. rather than two).3 Structural abnormalities When the chromosome's structure is altered. In a reciprocal translocation. segments from two different chromosomes have been exchanged. an entire chromosome has . 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. Tetrasomy.

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

New techniques were needed to definitively solve the problem: 1. Considering their techniques. at first favoring 46. the discoverer of mitosis. Their behavior in animal (salamander) cells was described by Walther Flemming. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. Pre-treating cells in a hypotonic solution.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). in contrast to their genic contents. which swells them and spreads the chromosomes . 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. Using cells in culture 2. in 1882. The name was coined by another German anatomist. 4.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. 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. von Waldeyer in 1888. concluding an XX/XO sex determination mechanism. He revised his opinion later from 46 to 48. these results were quite remarkable. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. and he correctly insisted on man having an XX/XY system. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia.

Rather interestingly. Arresting mitosis in metaphase by a solution of colchicine 4. McClintock discovered transposons. During her cytogenetic work. Using Painter's technique they studied the polytene . McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes). 4. 4. a find which eventually led to her Nobel Prize in 1983. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist.3.6. In 1931. the great apes have 48 chromosomes. Human chromosome 2 was formed by a merger of ancestral chromosomes. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. persimilis from wild populations in California and neighboring states. Squashing the preparation on the slide forcing the chromosomes into a single plane 5.6 Applications in biology 4.6.2 Natural populations of Drosophila In the 1930s. reducing the number.

as they would if selectively neutral. 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. All the flies look alike whatever inversions they carry: this is an example of a cryptic polymorphism. 4. This had the benefit of eliminating migration as a possible explanation of the results. 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. Stocks containing inversions at a known initial frequency can be maintained in controlled conditions. as with most polymorphisms. which enabled feeding. breeding and sampling whilst preventing escape. cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy. . Using a method invented by L'Heretier and Teissier. In some congenital disorders.7 Human abnormalities and medical applications In the event of procedures which allowed easy enumeration of chromosomes. discoveries were quickly made related to aberrant chromosomes or chromosome number. Lejeune discovered patients with Down syndrome had an extra copy of chromosome 21. Down syndrome is also referred to as trisomy 21. In 1959. 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. Dobzhansky bred populations in population cages.chromosomes and discovered that the wild populations were polymorphic for chromosomal inversions. Evidence rapidly accumulated to show that natural selection was responsible.

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

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

advances were made in molecular cytogenetics. 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.In the 1980s. 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.1 Karyotyping . Hybridizing them to chromosomal preparations using existing techniques came to be known as fluorescent in situ hybridization (FISH). cloned and studied in ever greater detail. CHAPTER 5 Techniques 5.

Routine chromosome analysis (Karyotyping) refers to analysis of metaphase chromosomes which have been banded using trypsin followed by Giemsa, Leishmanns, or a mixture of the two. This creates unique banding patterns on the chromosomes. The molecular mechanism and reason for these patterns is unknown, although it likely related to replication timing and chromatin packing. Several chromosome-banding techniques are used in cytogenetics laboratories. Quinacrine banding (Q-banding) was the first staining method used to produce specific banding patterns. This method requires a fluorescence microscope and is no longer as widely used as Giemsa banding (G-banding). Reverse banding (Rbanding) requires heat treatment and reverses the usual white and black pattern that is seen in G-bands and Q-bands. This method is particularly helpful for staining the distal ends of chromosomes. Other staining techniques include C-banding and nucleolar organizing region stains (NOR stains). These latter methods specifically stain certain portions of the chromosome. C-banding stains the constitutive heterochromatin, which usually lies near the centromere, and NOR staining highlights the satellites and stalks of acrocentric chromosomes. High-resolution banding involves the staining of chromosomes during prophase or early metaphase (prometaphase), before they reach maximal condensation. Because prophase and prometaphase chromosomes are more extended than metaphase chromosomes, the number of bands observable for all chromosomes increases from about 300 to 450 to as many as 800. This allows the detection of less obvious abnormalities usually not seen with conventional banding.

5.2 Slide preparation Cells from bone marrow, blood, amniotic fluid, cord blood, tumor, and tissues (including skin, umbilical cord, chorionic villi, liver, and many other organs) can be cultured using standard cell culture techniques in order to increase their number. A mitotic inhibitor (colchicine, colcemid) is then added to the culture. This stops cell division at mitosis which allows an increased yield of mitotic cells for analysis. The cells are then centrifuged and media and mitotic inhibitor are removed, and replaced with a hypotonic solution. This causes the white blood cells or fibroblasts to swell so that the chromosomes will spread when added to a slide as well as lyses the red blood cells. After the cells have been allowed to sit in hypotonic, Carnoy's fixative (3:1 methanol to glacial acetic acid) is added. This kills the cells and hardens the nuclei of the remaining white blood cells. The cells are generally fixed repeatedly to remove any debris or remaining red blood cells. The cell suspension is then dropped onto specimen slides. After aging the slides in an oven or waiting a few days they are ready for banding and analysis. 5.3 Analysis Analysis of banded chromosomes is done at a microscope by a clinical laboratory specialist in cytogenetics (CLSp(CG)). Generally 20 cells are analyzed which is enough to rule out mosaicism to an acceptable level. The results are summarized and given to a board-certified cytogeneticist for review, and to write an interpretation taking into account the patients previous history and other clinical findings. The results are then given out reported in an International System for Human Cytogenetic Nomenclature 2009 (ISCN2009). 5.4 Fluorescent in situ hybridization

Fluorescent in situ hybridization refers to using fluorescently labeled probe to hybridize to cytogenetic cell preparations. In addition to standard preparations FISH can also be performed on:
• • • • • • •

bone marrow smears blood smears paraffin embedded tissue preparations enzymatically dissociated tissue samples uncultured bone marrow uncultured amniocytes cytospin preparations

5.5 Slide preparation The slide is aged using a salt solution usually consisting of 2X SSC (salt, sodium citrate). The slides are then dehydrated in ethanol, and the probe mixture is added. The sample DNA and the probe DNA are then co-denatured using a heated plate and allowed to re-anneal for at least 4 hours. The slides are then washed to remove excess unbound probe, and counterstained with 4',6-Diamidino-2phenylindole (DAPI) or propidium iodide. 5.7 Analysis Analysis of FISH specimens is done by fluorescence microscopy by a clinical laboratory specialist in cytogenetics. For oncology generally a large number of interphase cells are scored in order to rule out low level residual disease,

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

6.1 Image Processing The image processing step aims at image contrast enhancement and compensation of geometric distortions observed in each chromosome not related with its intrinsic shape or size. specifying data types. It enables fast development and execution. 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. and distribute your MATLAB algorithms and applications. You can integrate your MATLAB code with other languages and applications. As a result. The MATLAB language supports the vector and matrix operations that are fundamental to engineering and scientific problems. With the MATLAB language. such as declaring variables. 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. one line of MATLAB code can often replace several lines of C or C++ code.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. MATLAB eliminates the need for ‘for’ loops. and allocating memory. . In many cases.

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

If you attempt to filter the indexed image. You can perform certain conversions just using MATLAB syntax. and the results might not be meaningful. 6. you must first convert it to true color format. MATLAB simply applies the filter to the indices in the indexed image matrix. so the image displays as shades of gray. listed in the following table.I.I.I). you can convert a grayscale image to true color format by concatenating three copies of the original matrix along the third dimension. RGB = cat (3.1 Image conversion The toolbox includes many functions that you can use to convert an image from one type to another. When you apply the filter to the true color image. there are other functions that return a different image type as part of the operation they perform. . if you want to filter a color image that is stored as an indexed image. For example. as is appropriate.3) Edge detection 4) Two dimensional convolutions. MATLAB filters the intensity values in the image. The resulting true color image has identical matrices for the red. green. and blue planes.2. the region of interest functions returns a binary image that you can use to mask an image for filtering or for other operations. For example. For example. In addition to these image type conversion functions.

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

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

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

end end if flag~=1 L_number(Index)=L(i.imy).31.49. end %h=figure.55. for x=1:46 [r.43.42.7.2).9.52.56.41.j).32.45.y1)=255.8.6. [sx sy]=size(rc).39.imshow(n1.15. .66].57.22.33.60.[]).54.59.20. n1=zeros(imx.26.11.29. rc = [r c].27.40. end flag=0.10.51. end end L_number.1).4. end.62. Index=Index+1.14.50.c] = find(L==L_number((Test_number(x)))).24. y1=rc(i. Test_number=[3.48.28.65. 36.35. n1(x1. for i=1:sx x1=rc(i.19.30.21.38.

skel=im2bw(skel. f=imcomplement(f).y)==1 Circumference_sum=Circumference_sum+1. [m n]=size(BW1).8).1).Inf). Arm_length_sum=0. Circumference_sum=0.'canny'). s1=bwmorph(s.'skel'.'spur'. Arm_length=zeros(46.1).end Circumference=zeros(46.'. Area=zeros(46. s=bwmorph(skel. for x=1:m for y=1:n if BW1(x. for i=1:46 f=imread(strcat(num2str(i).bmp')). skel=im2double(f). end end end Circumference(i)=Circumference_sum. BW=double(BW). BW=im2bw(f).1. BW1=edge(BW.1). .5*graythresh(skel)).

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

2)==46 Pair(46.1)=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(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)).1)=46. end end Pair. Pair(46. Pair(i.2)=j.Area.2)=i+1. Pair=zeros(46.2)=i.2). Pair(i. . Pair(i.1)=i. end end end for i=1:45 if Pair(i.

imshow(f2). figure_flag=1. flag=0. figure_flag=figure_flag+1. end f2=imread(strcat(num2str(Pair(i. end flag=0.2. imshow(f1).'. if figure_flag~=47 subplot(23.bmp')).1)==delete(j) flag=1. for i=1:46 for j=1:46 if Pair(i. . delete(figure_flag)=Pair(i. end f1=imread(strcat(num2str(Pair(i.1)).bmp')).2)). figure_flag=figure_flag+1.figure_flag).'.2.1).figure_flag).delete=zeros(46. end end if flag~=1 if figure_flag~=47 subplot(23.2).

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

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

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

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