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

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

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

In spite of their appearance. microtubules grow from centrosomes located at opposite ends of the cell and also attach to the centromere at specialized structures called kinetochores. This is the only natural context in which individual chromosomes are visible with an optical microscope. The microtubules then pull the chromatids apart toward the centrosomes. . q-g "grande").1 chromosome paired model Aneuploidy is usually caused by spindle fiber failure in meiosis I or II. one of which is present on each sister chromatid. They cease to function as accessible genetic material (transcription stops) and become a compact transportable form. 1. A special DNA base sequence in the region of the kinetochores provides. and they form the classic four arm structure. small) and the longer arms are called q arms (q follows p in the Latin alphabet. along with special proteins. chromosomes are structurally highly condensed.Fig 1.4 Metaphase chromatin and division In the early stages of mitosis or meiosis (cell division). longer-lasting attachment in this region. the chromatids are uncoiled and DNA can again be transcribed. Such a failure of the separation of homologous chromosomes or sister chromatids is called nondisjunction. a pair of sister chromatids attached to each other at the centromere. the chromatin strands become more and more condensed. During mitosis. The shorter arms are called p arms (from the French petit. so that each daughter cell inherits one set of chromatids. which enables these giant DNA structures to be contained within a cell nucleus. Once the cells have divided. This compact form makes the individual chromosomes visible.

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

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

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. The sister chromatids are held together by a specialized region of the chromosome known as the centromere. the period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs. . Each chromosome now has an identical copy of itself. the parent cell must make a copy of each chromosome before mitosis. These two cells are identical and do not differ in any way from the original parent cell. Because each resultant daughter cell should be genetically identical to the parent cell. and together the two are called sister chromatids. The genome is composed of a number of chromosomes—complexes of tightly-coiled DNA that contain genetic information vital for proper cell function.

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

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

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

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

This is called open mitosis. undergo a variation called closed mitosis where the spindle forms inside the nucleus. one attached at each chromatid.2. the motor activates. Although the kinetochore structure and function are not fully understood. such as algae or trichomonads. . kinetochore microtubules begin searching for kinetochores to attach to. When the spindle grows to sufficient length. since they are absent from plants. Each chromosome forms two kinetochores at the centromere. A kinetochore is a complex protein structure that is analogous to a ring for the microtubule hook.formation of the spindle. provides the pulling force necessary to later separate the chromosome's two chromatids. When a microtubule connects with the kinetochore. or its microtubules are able to penetrate an intact nuclear envelope. on an average 20 ). it is known that it contains some form of molecular motor. Prometaphase is sometimes considered part of prophase. using energy from ATP to "crawl" up the tube toward the originating centrosome. This motor activity. 2.2 Prometaphase The nuclear envelope disassembles and microtubules invade the nuclear space. Fungi and some protists. and it occurs in most multicellular organisms. it is the point where microtubules attach themselves to the chromosome ( about 1-40 in number. coupled with polymerisation and depolymerisation of microtubules. A number of nonkinetochore microtubules find and interact with corresponding nonkinetochore microtubules from the opposite centrosome to form the mitotic spindle. and centrosomes are not always used in mitosis.

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

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

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

.3 Cell replacement In some parts of body. skin and digestive tract. This is the basis of the development of a multicellular body from a single cell i.g. 2.2 Development and growth The number of cells within an organism increases by mitosis.1Significance Mitosis is important for the maintenance of the chromosomal set. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall. Following are the occasions in the lives of organism where mitosis happens: 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.division is also driven by vesicles derived from the Golgi apparatus.5. Similarly. New cells are formed by mitosis and so are exact copies of the cells being replaced. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell. 2. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis.5. The phragmoplast is a microtubule structure typical for higher plants.4 Regeneration .5. e. The end of cytokinesis marks the end of the M-phase. zygote and also the basis of the growth of a multicellular body.e. whereas some green algae use a phycoplast microtubule array during cytokinesis. separating the two nuclei. 2.5. which move along microtubules to the middle of the cell.

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

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

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

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

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

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

when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. concluding an XX/XO sex determination mechanism. New techniques were needed to definitively solve the problem: 1. 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. the discoverer of mitosis. The next stage took place after the development of genetics in the early 20th century. Considering their techniques. these results were quite remarkable. in contrast to their genic contents. Pretreating cells in a hypotonic solution. The name was coined by another German anatomist. which swells them and spreads the chromosomes . Using cells in culture 2.3. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. at first favoring 46. Painter in 1922 was not certain whether the diploid number of humans was 46 or 48. He revised his opinion later from 46 to 48. in 1882.1 History of karyotype studies Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. von Waldeyer in 1888. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. and he correctly insisted on humans having an XX/XY system.

It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes. such as Giemsa. Human chromosome 2 was formed by a merger of ancestral chromosomes. is applied after cells have been arrested during cell division by a solution of colchicine. Usually. white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture. 3. The sex of an unborn fetus can be determined by observation of interphase cells.2 Observations Six different characteristics of karyotypes are usually observed and compared: . Arresting mitosis in metaphase by a solution of colchicines 4.2. For humans. reducing the number. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. a suitable dye.3.2.2 Observations on karyotypes 3. [16] Sometimes observations may be made on non-dividing (interphase) cells. the great apes have 48 chromosomes. Cutting up a photomicrograph and arranging the result into an indisputable karyogram.1 Staining The study of karyotypes is made possible by staining. Rather interestingly. 3.

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

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

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

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

Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants. and aneuploids are another example. of a karyotype is the number of visible major chromosomal arms per set of chromosomes.means that chromosome number can vary even within one interbreeding population. 3. occurs mainly in plants. Polyploidy in animals is much less common. the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present. Haplo-diploidy. where one sex is diploid. 15.3. though in this case they would not be regarded as normal members of the population. 14. FN ≤ 2n. It is a common arrangement in the Hymenoptera. Polyploidy. Polyploidy in lower plants (ferns. and the other haploid. It has been of major significance in plant evolution according to Stebbins. Thus. but in grasses the average is much higher. FN. where there are more than two sets of homologous chromosomes in the cells. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%.4 Ploidy Ploidy is the number of complete sets of chromosomes in a cell.Endopolyploidy occurs when in adult differentiated . 21 and 22). 3. and in some other groups. due to the presence of five acrocentric chromosome pairs (13. about 70%. horsetails and psilotales) is also common. but it has been significant in some groups.3 Fundamental number The fundamental number. Humans have FN = 82.

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

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. where the gametic (= haploid) numbers form the series x = 3. that the two chromosome morphs are adapted to different habitats. living from rainforests to . There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast. where every number from x = 3 to x = 15 is represented by at least one species. 3. and Crocus. 3. 6. When this happens. the great apes have 24x2 chromosomes whereas humans have 23x2. 4.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. some mantids of the genus Ameles. the European shrew Sorex araneus.000 km2). Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups.Aneuploidy may also occur within a group of closely related species. 5. In about 6. Human chromosome 2 was formed by a merger of ancestral chromosomes.500 sq mi (17. Classic examples in plants are the genus Crepis. the chromosome number is variable from one individual to another. [41] Closer to home. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world. and 7. Well-researched examples are the ladybird beetle Chilocorus stigma. reducing the number.

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

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

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

8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale. The critical region for this syndrome is deletion of 15.2) 3. which is written as 46.del(5)(p15. 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. Image processing software then assigns a pseudo color to each spectrally different combination.7. Because there are a limited number of spectrally-distinct fluorophores. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope. It is written as 46. allowing the visualization of the individually colored chromosomes.5p-. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores.XX. Short sequences of DNA from specific loci all over the genome are isolated and enumerated.XX. 3.2.chromosome 5. .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. This method is also known as virtual karyotyping.

CHAPTER 4 .

as in the presence of extra or missing chromosomes. the most common male chromosomal disease. otherwise known as 47. Chromosomal abnormalities that lead to disease in humans include • • Turner syndrome results from a single X chromosome (45. including . Klinefelter syndrome. a common chromosomal disease. • • Edwards syndrome is caused by trisomy (three copies) of chromosome 18. trisomies. Also documented are trisomy 8. often occur as a result of nondisjunction during meiosis in the formation of a gamete. are common numerical abnormalities. or structural. is caused by trisomy of chromosome 21. although they generally do not survive to birth. as in derivative chromosome. X or 45. translocations. Structural abnormalities often arise from errors in homologous recombination. • • Patau syndrome is caused by trisomy of chromosome 13. trisomy 9 and trisomy 16. inversions. in which three copies of a chromosome are present instead of the usual two. or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells. Down syndrome. Numerical abnormalities. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body.4.1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical. X0). Some disorders arise from loss of just a piece of one chromosome. large-scale deletions or duplications. also known as aneuploidy. XXY is caused by an extra X chromosome.

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

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

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

Painter in 1922 was not certain whether the diploid number of man was 46 or 48. New techniques were needed to definitively solve the problem: 1. The name was coined by another German anatomist. He revised his opinion later from 46 to 48. The next stage took place after the development of genetics in the early 20th century. and he correctly insisted on man having an XX/XY system. Their behavior in animal (salamander) cells was described by Walther Flemming. Pre-treating cells in a hypotonic solution. Considering their techniques. 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. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. Investigation into the human karyotype took many years to settle the most basic question: how many chromosomes does a normal diploid human cell contain? In 1912. in 1882. Using cells in culture 2. in contrast to their genic contents. the discoverer of mitosis. at first favoring 46.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. 4.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). which swells them and spreads the chromosomes . von Waldeyer in 1888. these results were quite remarkable.

McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize. 4. a find which eventually led to her Nobel Prize in 1983. Rather interestingly.6 Applications in biology 4.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. persimilis from wild populations in California and neighboring states.6. reducing the number. Squashing the preparation on the slide forcing the chromosomes into a single plane 5.6. Cutting up a photomicrograph and arranging the result into an indisputable karyogram.2 Natural populations of Drosophila In the 1930s. Using Painter's technique they studied the polytene . Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes. Human chromosome 2 was formed by a merger of ancestral chromosomes. McClintock discovered transposons. the great apes have 48 chromosomes. In 1931. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes). 4. Arresting mitosis in metaphase by a solution of colchicine 4.3. During her cytogenetic work.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

22.60. end flag=0.27.14.32.26.15.7. [sx sy]=size(rc).50.49.38.imy).20.48.66].19. for x=1:46 [r.21.62. end end L_number.55.1).end end if flag~=1 L_number(Index)=L(i. y1=rc(i.59.65.11.10. end %h=figure.41. end.c] = find(L==L_number((Test_number(x)))).39. n1=zeros(imx.2).45.31.9.4.52. Test_number=[3.j). for i=1:sx x1=rc(i.43.54.[]). n1(x1.57. 36.24.42.40.y1)=255. .28. Index=Index+1.8.29.imshow(n1.30.6.35. rc = [r c].56.33.51.

y)==1 Circumference_sum=Circumference_sum+1. end end end Circumference(i)=Circumference_sum.bmp')). BW=double(BW).'canny'). f=imcomplement(f).end Circumference=zeros(46. BW1=edge(BW. s1=bwmorph(s.5*graythresh(skel)). Arm_length_sum=0. for x=1:m for y=1:n if BW1(x. Circumference_sum=0.8).1). Arm_length=zeros(46.1. [m n]=size(BW1).1). s=bwmorph(skel. skel=im2double(f). skel=im2bw(skel. for i=1:46 f=imread(strcat(num2str(i).'spur'.Inf).1). BW=im2bw(f). . Area=zeros(46.'.'skel'.

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

2)=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)=i. end end end for i=1:45 if Pair(i.2)==46 Pair(46. Pair(46. 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)). end end Pair. Pair(i. Pair=zeros(46. Pair(i.2).Area.1)=i. .2)=i+1.1)=46.2)=j.

figure_flag=figure_flag+1. end end if flag~=1 if figure_flag~=47 subplot(23.'. figure_flag=figure_flag+1.1)==delete(j) flag=1.2. end f2=imread(strcat(num2str(Pair(i.bmp')). imshow(f2).1)). if figure_flag~=47 subplot(23.figure_flag). figure_flag=1.'. .2).2)). end flag=0. for i=1:46 for j=1:46 if Pair(i.bmp')). end f1=imread(strcat(num2str(Pair(i. imshow(f1).figure_flag). delete(figure_flag)=Pair(i.delete=zeros(46.1). flag=0.2.

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

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

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

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